JPH06209736A - Production of monomer curculin and taste modifier containing monomer curculin - Google Patents

Abstract

PURPOSE:To obtain a production method of a taste-modifying agent having good absorption to the tongue and strong taste modifying activity and sweetness activity and a taste-modifier containing monomer curculin having good absorption to the tongue and strong taste modifying activity and sweetness activity. CONSTITUTION:Monomer curculin is produced by treating dimer curculin with a disulfide bond reducing agent. The taste modifier contains monomer curculin.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a monomer curculin having enhanced taste-modifying activity and sweetness activity (taste-modifying effect), and a taste-modifying agent containing the monomer curculin obtained by the method. It is a thing.

[0002]

2. Description of the Related Art The applicant has previously proposed the protein curculin
A method of extracting from a fruit of Curculigo latifolia or a dried product thereof with an aqueous solution of a salt having a concentration of 0.01 M or more has been found (JP-A-2-104).
263).

[0003]

However, the protein curculin obtained by the method described in JP-A-2-104263 has a poor adsorption effect on the tongue.

The present applicant has applied for a taste-modifying substance consisting of a curculin-like substance (Japanese Patent Application No. 3-2539).
No. 14), this curculin-like substance forms a dimer by an intrachain bond and an interchain bond.

Therefore, a first object of the present invention is to provide a method for producing a monomer curculin which is well adsorbed to the tongue and has a strong taste-modifying activity and strong sweetening activity. A second object of the present invention is to provide a taste modifier containing monomer curculin, which is well adsorbed to the tongue and has a strong taste modifying activity or strong sweetening activity.

[0006]

[Means for Solving the Problems] As a result of diligent research on curculin in order to achieve the above-mentioned object, the present inventors found that curculin is active even in a monomer state, and that monomer curculin exists naturally in fruits and the like. It was found that the taste-modifying activity and the sweetening activity are stronger than those of Daimark Luculin.

The present invention has been made based on the above findings, and provides a method for producing monomer curculin having an enhanced taste-modifying effect, which comprises treating dimarkluculin with a disulfide bond reducing agent. is there. The present invention also provides a taste modifier containing the monomer curculin obtained by the above-mentioned production method of the present invention.

First, the method for producing the monomer curculin of the present invention will be described in detail below.

The dimark ruculin used in the present invention was obtained by a biological method utilizing the fruit of Curculigo latifolia or a processed product thereof, the dimark ruculin obtained from them, and further the curculin gene. An example is Daimark Lucrin.

As the above-mentioned fruits or processed products thereof, since the fruit skins and seeds of Curculigo latifolia do not contain dimarkcruclin, raw fruits of Curculigo latifolia, excluding skins and seeds, are dried or frozen. Examples thereof include fruits, and treated fruits obtained by subjecting these to crushing or crushing.

As a method for drying the fruits, drying in a relatively low temperature range such as shade drying, low temperature drying, freeze drying and vacuum drying is preferable, but it is not particularly limited. The method of freezing the fruit may be slow freezing, quick freezing, self freezing, etc., and is not particularly limited. Also, the method of crushing or breaking is not particularly limited.

[0012] Dimark cuculin obtained from the fruit of Curculigo latifolia or a processed product thereof is a curculin extracted from the fruit or a processed product thereof, for example,
Curculin extracted by the extraction method described in JP-A-2-104263, that is, curculin extracted from the fruit or a dried product thereof with an aqueous solution of a salt having a concentration of 0.01 M or more, or Japanese Patent Application No. 4-317533. Curculin extracted by the extraction method described in No. 1, ie, the fruit or a processed product thereof, and an aqueous acid solution are added to the mixture to adjust the pH to 3
Examples of curculin extracted include the following, but the method for extracting curculin is not limited thereto.

Further, the degree of purification of dimarkuculin obtained by the above extraction method is not limited. That is, salting out with ammonium sulfate, sodium sulfate, potassium phosphate, magnesium sulfate, sodium citrate, sodium chloride, etc., solvent precipitation with a polar solvent such as methanol, ethanol, isopropanol, acetone, C
It may be purified by a known protein purification method by chromatography such as ion exchange chromatography such as M-Sepharose, Amberlite, IRC-50 and molecular sieve chromatography such as Sephadex G-100.

In the present invention, the high-purity dimark ruculin is one which has been subjected to ion exchange chromatography, molecular sieve chromatography and salting-out treatment.

Thus, the production method of the present invention is to produce a monomer curculin by treating the above-mentioned dimarkcruclin having a disulfide bond with a reducing agent. Here, the reducing agent treatment of the present invention refers to a treatment of reacting a reducing agent with a thiol group in a dimarkluculin molecule to cleave a disulfide bond.

The disulfide bond reducing agent used in the present invention may be any reducing agent capable of exchanging with a thiol group, and specifically, dithiothreitol, dithioerythritol, mercaptoethanol, reduced glutathione, Although cysteine and the like can be mentioned, reduced glutathione and cysteine are particularly preferable from the viewpoint of taste and safety.

The above-mentioned reducing agent may be added in an amount necessary for reducing dimarkuclucrin into a monomer. For example, reduced glutathione or cysteine is 1 mM or more, preferably 15 mM or more, with respect to dimark ruculin (high-purity dimark ruculin content 100 μg / ml).
More preferably, it may be added at a concentration of 50 mM or more. In addition, dithiothreitol is used for dimark ruculin (high-purity dimark thruculin content 100 μg /
ml) to 0.25 mM or more, preferably 1 mM or more, more preferably 2 mM or more. Further, dithioerythritol and mercaptoethanol may be added in the same amount as that of dithiothreitol.

However, since the reducing agent does not always have a preferable taste and also has a protein denaturing action, it is preferable to use the necessary minimum amount according to the purpose of use.

The treatment temperature for the treatment with the disulfide bond reducing agent may be room temperature or a heating temperature to the extent that the dimark cruculin or the monomer curculin is not denatured, specifically 20 to 100 ° C. Since the reaction time when treated with the agent is reduced relatively quickly, 1 minute or less is sufficient at 60 ° C or higher, and 1 minute or less is required at 60 ° C or lower, but the reaction time is longer than necessary. I don't mind.

The obtained monomer curculin can be suitably used as a taste-modifying agent and as a sweet protein, and the taste-modifying activity and sweetening activity of the monomer curculin are the same as those of the conventional Dimarkcruclin. The amount used for obtaining taste-modifying activity and sweetening activity is about 1/1.
It can be 5 to 1/5. Furthermore, the monomer curculin has a sweetness duration of about 1.5 to 3 times longer than that of dimarkcruclin.

Next, the taste modifier containing the monomer curculin obtained by the above production method of the present invention will be described.

In the taste modifier of the present invention, the monomer curculin is converted into high-purity dimarkcruclin, preferably 0.02 to 700 ppm, more preferably 0.2 to 7 ppm.
Contains 0 ppm.

The monomer curculin contained in the taste-modifying agent of the present invention may be a stabilized monomer curculin obtained by modifying a free thiol group in the monomer curculin. At this time, as the modifier used for modifying the thiol group of the monomer curculin,
Those generally used for modification of thiol groups of proteins can be mentioned. Specifically, monoiodoacetic acid (carboxymethylated), iodoacetamide (carboxamidomethylated), ethyleneimine (aminoethylated), 2-bromoethylamine (aminoethylated), 4-vinylpyridine (pyridylethylated), acrylonitrile. (Cyanoethylation) and the like can be mentioned, with preference given to iodoacetamide.

The free thiol group in the monomer curculin is modified by reacting the thiol group modifying agent with the SH group.

The monomer curculin obtained by the production method of the present invention and the taste-modifying agent of the present invention containing the monomer curculin may be taken as they are, or may be appropriately mixed with foods, beverages or pharmaceuticals before use. May be. When used by being mixed with foods, beverages, drugs, etc., it can be determined according to the purpose and application by referring to the taste-modifying activity and sweetening activity of the monomer curculin, but it can be determined depending on the intended use. In terms of conversion, it is preferable to contain 0.02 to 700 ppm, particularly 0.2 to 70 ppm.

Further, the monomer curculin obtained by the production method of the present invention and the taste modifier of the present invention containing the monomer curculin are processed into powder, solution, sheet, spray or emulsion. Can, food,
When used by being mixed with a beverage, a drug or the like, it may be processed according to their properties.

[0027]

The monomer curculin obtained by the production method of the present invention and the taste modifier of the present invention containing the monomer curculin include a sour substance or water containing the monomer curculin or the taste modifier in the mouth. If you eat
Alternatively, when the monomer curculin or the taste-modifying agent is eaten with a sour substance or water, it has an effect of acting on the receptor membrane of the tongue to make it taste sweet. In addition, if a food or the like containing the above-mentioned monomer curculin or the above taste-modifying agent is eaten in advance, sweetness can be felt when eating a sour substance or water.

[0028]

EXAMPLES Next, the present invention will be specifically described by showing Examples.

Reference Example 1 (Preparation of Dimark Luculin) 30 g of flesh of Curculigo latifolia was taken, 40 ml of water was added thereto, and the mixture was homogenized and centrifuged (12,
500 rpm, 60 minutes). This supernatant was brown and had no taste-modifying activity. 40 ml of water was added to the obtained precipitate and homogenized, followed by centrifugation (12,500 r).
pm, 20 minutes). The supernatant was colorless and had no taste modifying activity.

Next, 40 ml of 0.5 M sodium chloride solution was added to the obtained precipitate, and the mixture was homogenized and centrifuged (30,000 rpm, 60 minutes). The resulting supernatant was colorless and a crude curculin (dimer) extract having taste-modifying activity was obtained. Furthermore, the extraction operation with 40 ml of 0.5 M sodium chloride solution was repeated twice, and these three extracts were combined.

80 parts of this crude curculin extract (three times)
Ammonium sulphate was added to precipitate the active substance so that it was% saturated. Centrifuge (32,000 rpm, 60
The precipitate obtained by (1 min) was dissolved in 10 ml of water and freeze-dried to obtain 30.5 mg of crude curculin (dimer).

Next, 100 ml of crudely purified curculin was added to 10
Dissolved in mM phosphate buffer (pH 6.8), CM-Sepharose CL-6B column (diameter 2.2 cm x length 18 c)
m, 68 ml, manufactured by Pharmacia LKB Biotechnology) and adsorbed. Subsequently, the flow-through fraction was washed with 0.01 M phosphate buffer (pH 6.8), and then curculin was eluted by a linear concentration gradient elution method of a sodium chloride solution of 0 to 1.0 M (flow rate 5 ml / hour, 1 fraction 5 ml, total eluate volume 500 ml). Eluted protein was monitored by absorption at 280 nm. As a result, a fraction containing the taste modifier curculin was eluted at a sodium chloride concentration of around 0.4M.

Ammonium sulfate was added to this fraction containing curculin so as to be 80% saturated to precipitate the active substance. The precipitate obtained by centrifugation (32,000 rpm, 60 minutes) was dissolved in 1.5 ml of 0.01 M phosphate buffer (pH 6.8). This concentrate is Sephadex (Pharmacia LKB Biotechnology) G
-100 column (diameter 1.6 cm x length 58 cm, 160 m
l) and separated with 0.01 M phosphate buffer (pH 6.8) containing 0.5 M sodium chloride (flow rate 8.4).
ml / hour, 1 fraction 2.8 ml, total elution amount 182 ml). Protein was monitored by absorption at 280 nm. as a result,
The peak of the taste modifier curculin was eluted.

Ammonium sulfate was added to this peak fraction to 80% saturation to precipitate the active substance.
The precipitate obtained by centrifugation (32,000 rpm, 60 minutes) was dissolved in 1.5 ml of 2M ammonium acetate solution. This solution was treated with a Sephadex G-25 (9.1 ml) column equilibrated with a 2M ammonium acetate solution, and then freeze-dried to obtain 8.6 mg of high-purity dimarklucrin.

Subsequently, the molecular weight of the obtained high-purity dimarkcruclin was analyzed by electrophoresis. That is, a sample solution for electrophoresis was prepared by using the high-purity dimarkuculin obtained above as a sample solution having a concentration of 0.1 mg / ml. This sample solution for electrophoresis was subjected to polyacrylamide gel electrophoresis according to the method of Laemmli (Laemmli, UK, Nature, 227, 680, 1970) by 10 μl each. The gel after electrophoresis was stained with Coomassie Brilliant Blue R-250, and after decolorization, the molecular weight was quantified by comparing the mobility of the marker protein with the sample. As a result, the molecular weight of the above-mentioned high-purity Dymark ruculin was 23,800.

Example 1 (Treatment with Reduced Glutathione) 2.5 mg of high-purity dimarkluculin prepared in Reference Example 1
Was dissolved in 2.5 ml of 62.5 mM reduced glutathione solution and left at room temperature for 10 minutes to prepare a curculin solution treated with a reducing agent.

In order to confirm that the curculin in the reducing agent-treated curculin solution is a monomer curculin,
30 μl of this solution was added to a microcentrifuge tube (1.5 ml volume).
20 μm of Tris-HCl buffer (pH 6.8) containing 2.5% sodium dodecyl sulfate, 25% glycerol and 25 ppm bromophenol blue.
1 was added and stirred to prepare an electrophoretic sample. This sample 2
When μl was analyzed in the same manner as in the method of Reference Example 1, a band was confirmed at the position of molecular weight 13,800.

The curculin solution 20 treated with the reducing agent is also used.
When 0 μl was diluted with 50 mM phosphate buffer (pH 6.0) to 3 ml and contained in the mouth to measure the taste-modifying activity, the sweetness was 0.34 M sucrose solution, and This sweetness lasted for about one and a half hours.

Comparative Example 1 2.5 ml of a 62.5 mM reduced glutathione solution used in Example 1 was replaced with an aqueous solution of phosphoric acid (pH 2.5) 2.5.
A curculin solution treated with an aqueous phosphoric acid solution was prepared in the same manner as in Example 1 except that ml was used.

The curculin in the curculin solution treated with the phosphoric acid aqueous solution was subjected to molecular weight measurement in the same manner as in Example 1. As a result, the molecular weight was 23,800, and monomer curculin was not obtained.

The taste-modifying activity of the above-mentioned phosphoric acid aqueous solution-treated curculin solution was measured in the same manner as in Example 1. As a result, the sweetness was sweetness equivalent to that of a 0.27 M sucrose solution, and This sweetness lasted for about 30 minutes.

Test Example 1 (Method for measuring taste-modifying activity of monomer curculin) Curcumin solution (5) treated with a reducing agent obtained in Example 1
3 ml of 0 mM phosphate buffer, pH 6.0 was included in the mouth for 3 minutes, and then the mouth was rinsed gently. Next, 3 ml of a 0.1 M citric acid solution was tasted, and the sweetness sensed at this time was converted into the sweetness of sucrose (molar concentration of sucrose) to obtain a taste-modifying activity (sweetness). However, in measuring sweetness activity, individual differences and variations in measurement were observed, so the average value of 10 panelists was used as the degree of sweetness. The results are as shown in [Table 1] below. The same test was carried out on the phosphoric acid aqueous solution-treated curculin solution obtained in Comparative Example 1, and the results are shown in [Table 1] below. As is clear from the results shown in [Table 1] below, the amount of monomer curculin used was 1/2 or less when the sweetness was the same as that of Dimarkcruclin.

[0043]

[Table 1]

Example 2 (Treatment with cysteine) Instead of 2.5 ml of the 62.5 mM reduced glutathione solution used in Example 1, a 62.5 mM cysteine solution 2.5 was used.
A curcumin solution treated with a reducing agent was prepared in the same manner as in Example 1 except that ml was used.

The reducing agent-treated curculin solution was measured for molecular weight in the same manner as in Example 1. As a result, the molecular weight was 13,8.
It was 00. That is, the curculin in the solution was a monomer curculin.

The taste-modifying activity of the curcumin solution treated with the reducing agent was measured in the same manner as in Example 1. The sweetness was 0.33 M, which was equivalent to that of the sucrose solution. The sweetness lasted for about one and a half hours.

Example 3 (Treatment with 2-mercaptoethanol) The same as Example 1 except that 2.5 ml of the 62.5 mM reduced glutathione solution used in Example 1 was replaced with 2.5 ml of a 2 mM 2-mercaptoethanol aqueous solution. A curcumin solution treated with a reducing agent was prepared in the same manner.

The curcumin solution treated with the reducing agent was measured for molecular weight in the same manner as in Example 1. The molecular weight was 13,8.
The curculin in the solution was monomeric curculin.

Example 4 (Treatment with dithiothreitol) The same as Example 1 except that 2.5 ml of a 22.5 mM dithiothreitol aqueous solution was used instead of 2.5 ml of the 62.5 mM reduced glutathione solution used in Example 1. Then, a curcumin solution treated with a reducing agent was prepared.

The reducing agent-treated curculin solution was measured for molecular weight in the same manner as in Example 1. As a result, the molecular weight was 13,8.
The curculin in the solution was monomeric curculin.

Comparative Examples 2 to 4 (Reaction with Organic Acid Having Reducing Power) 2.5 ml of 62.5 mM reduced glutathione solution used in Example 1 was replaced with 2.5 ml of 500 mM L-ascorbic acid (Comparative Example). 2), 500 mM DL-lactic acid 2.5
ml (Comparative example 3), 2.5 ml of 500 mM DL-malic acid
An organic acid-treated curculin solution having reducing power was prepared in the same manner as in Example 1 except that (Comparative Example 4) was used.

The organic acid-treated curculin solution having reducing power was measured for molecular weight in the same manner as in Example 1. As a result, the molecular weight was 23,800, and no monomer curculin was formed from this treatment.

Example 5 (Modification of thiol group) To 2.5 ml of a monomer curculin solution prepared in the same manner as in Example 2, 43 mg of iodoacetamide (1.5 times the mole number of cysteine) was added, and the mixture was stirred at room temperature. After carrying out a carboxamide methylation reaction of a thiol group for 10 minutes, the mixture was allowed to stand at 0 ° C. for 1 hour to further advance the carboxamide methylation reaction. After standing, Sephadex G-25 was used to add ammonium bicarbonate buffer solution containing 2M urea and 2mM EDTA (p.
The solvent was exchanged for H8.0). When the molecular weight of this thiol group-modified monomer curculin solution was measured in the same manner as in Example 1, the molecular weight was 13,800.

Subsequently, 2 ml of the obtained thiol group-modified monomer curculin solution was dialyzed overnight against 2 liters of 0.05 M phosphate buffer (pH 6.0) containing 0.5 M sodium chloride. The obtained dialysate was analyzed for molecular weight in the same manner as in Reference Example 1. As a result, a band was confirmed at the molecular weight of monomer curculin at 13,800, and it was confirmed that the curcumin modified with a thiol group was stable. It was

Example 6 (Monomerization of crudely purified curculin) 2.5 mg of the high-purity dimarked cruculin used in Example 1
In place of the crude purified curculin 6.2 prepared in Reference Example 1.
Example 1 was repeated except that 5 mg was used.
The molecular weight was 13,800, and crude monomer crude curculin was obtained.

About this monomer crudely purified curculin,
When the taste-modifying activity was measured in the same manner as in Example 1, the sweetness was found to be sweetness equivalent to that of a 0.32 M sucrose solution, and this sweetness lasted for about one and a half hours.

Comparative Example 5 In place of 2.5 mg of high-purity dimarkcruculin used in Comparative Example 1, 6.25 mg of crudely purified curculin used in Reference Example 1
Was carried out in the same manner as in Comparative Example 1 except that was used, and the molecular weight was 23,800, and crude monomer crude curculin could not be obtained.

The taste-modifying activity of this curculin was measured in the same manner as in Example 1. As a result, the sweetness was sweetness equivalent to that of a 0.26 M sucrose solution, and the sweetness was about 30 minutes. Lasted.

Examples 7 to 8 and Comparative Examples 6 to 7 Lemon juice jelly having a composition of 7.5% lemon juice, 2% gelatin and the balance water was produced. On the other hand, in 7.5% lemon juice, the monomer curculin solutions obtained in Example 1 (using high-purity curculin) and Example 6 (using crude purified curculin) (Examples 7 and 8 respectively) and Comparative Example 1 ( High-purity curculin was used) and the dimark rucurin solutions obtained in Comparative Example 5 (crudely purified curculin) (Comparative Examples 6 and 7, respectively) were added in an amount of 2% to produce curculin-containing lemon juice.

Next, after eating each curculin-containing lemon juice and eating the above-mentioned lemon juice jelly, it was found that when the curcurin-containing lemon juice of Example 7 (using a monomer high-purity curculin solution) was eaten The lemon juice jelly was found to have a stronger sweetness than when the curculin-containing lemon juice of Comparative Example 6 (using a dimer high-purity curculin solution) was eaten. Also, similarly, Example 8
The curcumin-containing lemon juice (using the monomer crude refined curculin solution) was compared with the curculin-containing lemon juice of Comparative Example 7 (using the dimer crude refined curculin solution) in the lemon juice. I felt a strong sweetness in the jelly.

Example 9 To 10 ml of the crude curculin solution obtained by the first extraction operation with the 0.5 M sodium chloride solution of Reference Example 1, 3
A curcumin solution treated with a reducing agent was prepared in the same manner as in Example 1 except that 20 μl of a 2M reduced glutathione solution was added.

To confirm the molecular weight of curculin in the reducing agent-treated curculin solution, 30 μl of the reducing agent-treated curculin solution was added to a microcentrifuge tube (1.5 ml volume).
20 μm of Tris-HCl buffer (pH 6.8) containing 2.5% sodium dodecyl sulfite, 25% glycerol and 25 ppm bromophenol blue.
After adding 1 and stirring, analysis was performed by the same method as in Reference Example 1. As a result, a band was confirmed at 13,800, and a crude monomer curculin solution was obtained.

Next, the taste-modifying activity of 3 ml of the crude monomeric curculin solution obtained above was measured in the same manner as in Example 1. The sweetness was 0.34 M, which was equivalent to that of a sucrose solution. The sweetness lasted for about one and a half hours.

[0064]

EFFECTS OF THE INVENTION According to the production method of the present invention, monomer curculin which is well adsorbed on the tongue and has a strong taste-modifying activity or sweetness activity and which is suitably used as a taste-modifying agent or a sweet protein can be obtained. Further, the taste-modifying agent of the present invention has a strong taste-modifying activity even in a small amount because it has good adsorption to the tongue.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Hiroshi Sugiyama 7-35 Higashiohisa, Arakawa-ku, Tokyo Within Asahi Denka Kogyo Co., Ltd. (72) Hiroshige Kono 7-2-35 Higashiohisa, Arakawa-ku, Tokyo No. Asahi Denka Kogyo Co., Ltd. (72) Inventor Yoshie Kurihara 7-4-7 Okusawa, Setagaya-ku, Tokyo

Claims (2)

[Claims] 1. A method for producing a monomer curculin having an enhanced taste-modifying effect, which comprises treating dimarkcruclin with a disulfide bond reducing agent. 2. A taste modifier containing a monomer curculin obtained by the production method according to claim 1.