JPH02104263A - Protein curculine and use thereof - Google Patents

Abstract

PURPOSE:To obtain a taste-modifying substance having high stability by extracting fruit or dried fruit of Curculigo latifolia with an aqueous solution of a salt having a specific concentration. CONSTITUTION:The objective protein curculine is produced by extracting fruit or dried fruit of Curculigo latifolia with an aqueous solution of a salt having a concentration of >=0.01M. The dried fruit of Curculigo latifolia is e.g., a sun- dried material, hot-air dried material, freeze-dried material such as freeze-dried pulp, etc. The extraction of curculline is insufficient when the salt concentration of the extraction solution is <0.01M. On the contrary, too high concentration of salt increases the labor of the desalting operation after extraction and, accordingly, the salt concentration of the aqueous solution is preferably 0.1-1.0M to attain balanced extraction efficiency and the purification labor after the extraction.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel protein curculin and its use as a taste modifier.

(Prior art) Proboscis'ff (which acts on the receptor membrane of the tongue and changes the taste of food)
As a taste modifier, it has traditionally been contained in the leaves of Gymnema sylvestre (Gymnema sylvestre) as a substance that makes the sweet taste less confusing when eaten during the day, or when eaten with a sweet substance or together with a sweet substance. gymnemic acid, and jujube (Ziziphus juj)
It is known that didyfin is contained in the leaves of the miracle fruit (Synsepul
Miraculin contained in the fruit of M. dulcificu+g) is known.

Also, Curculig.

1atifolia) is a plant of the genus Atifolia in the family Asteraceae that grows naturally in West Malaysia, southern Thailand, etc., and its fruit is edible and is known to have an appetite-stimulating effect.

[Problem to be solved by the invention]

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.

Furthermore, the fruits of Curculigo latifolia are not known to have any properties other than those mentioned above.

Therefore, an object of the present invention is to provide a novel taste modifier with a high degree of stabilization.

[Means to solve the problem]

The present invention "Ff et al. discovered that if you eat a sour substance or water after eating the fruit of Curculigo latifolia, you will experience a sweet taste, and as a result of intensive research to find out what the trigger substance is, the fruits of Curculigo latifolia. (Frui
It has been found that a specific protein (named curculin) contained in t) is an inducing substance that deceives the taste of sweetness and achieves the above purpose.

The present invention was made based on the above-mentioned findings, and it is possible to obtain O,01 from Curculigo latifolia fruit or its dried product.
The present invention provides a protein curculin obtained by extraction with an aqueous salt solution having a concentration of M or higher, a taste modifier comprising curculin, a food, drink, or drug containing a taste modifier comprising curculin, and a method for extracting the protein curculin. It is.

Hereinafter, the protein curculin of the present invention will be explained in detail.

The curculin of the present invention is Curculigo latifolia (C
urcultgo latifolia) or its dried product by extraction with an aqueous salt solution having a concentration of 0.01 M or more.

The above salts include sodium, potassium, calcium, magnesium or ammonium hydrochloride, sodium, potassium, magnesium or ammonium phosphate, sodium, potassium, magnesium or ammonium carbonate, sodium, magnesium,
Calcium or ammonium sulfates or sulfites, sodium or potassium nitrates or nitrites,
Sodium or calcium lactate, alum,
Burnt alum, sodium acetate, sodium or potassium birophosphate, sodium or calcium propionate, sodium benzoate, sodium fumarate, sodium polyacrylate, etc. are used.

Further, the drying method of the fruit of Curculigo latifolia is not particularly limited, and sun-dried products, hot air-dried products, freeze-dried products such as freeze-dried bulbs, etc. can be used.

A typical example of a method for extracting curculin using an aqueous solution of the above salt is as follows.

The fruit of Curculigo latifolia or its dried product is homogenized with an aqueous solution of the above salt, and then filtered.
Curculin can be extracted by centrifugation, etc., but since curculin is contained in the water-insoluble part of the fruit pulp of Curculigo latifolia, add water to the fruit or its dried product, homogenize it, and wash thoroughly with water to extract the water-soluble part. It is preferable to extract the remaining aroma with an aqueous solution of the above-mentioned salt after removing the curculin.

If the aqueous salt solution used for extraction has a concentration of less than 0001M, curculin cannot be extracted sufficiently, so an aqueous salt solution with a concentration of 0.01M or more is required.

On the other hand, if the concentration is too high, desalting after extraction takes time and effort, so an aqueous salt solution with a concentration of 0.1 to 1.0 M is preferable from the standpoint of extraction efficiency and time and effort required for purification after extraction.

By desalting and drying the extract using an aqueous solution of the above-mentioned salt, a substance containing curculin that can be used for practical purposes can be obtained. However, the extract is further purified and purified by ion exchange with CM-Sepharose and HPLC using a gel column. By doing so, the purity can be increased, and then it is desalted and dried to obtain pure curculin. Of course, in addition to the above-mentioned purification methods, the purity of curculin can also be increased by fractionating the extract by salting out, solvent precipitation, or other known protein purification methods.

A specific example of the curculin of the present invention obtained in this manner has a molecular weight of about 12,500 DalLon (dalLon).
), the number of amino acid residues is 97, and the isoelectric point is 7.1, and this protein has a molecular weight of approximately 26,000 Daltons (d
Alton exists as a dimer. In addition, the amino acid composition of the above protein is as shown in Table 1 below, and the above protein contains a relatively large amount of aspartic acid,
Amino acid composition of leucine and glycine in Table 1 The protein curculin of the present invention described above is suitably used as a taste modifier.

The taste modifier of the present invention consisting of curculin may be ingested as it is, but the amount to be added to foods, etc., which can be appropriately mixed into foods, drinks, medicines, etc., is as follows:
It can be determined depending on the purpose and use with reference to the sweet taste-producing activity of curculin, which will be described later.

Furthermore, foods containing the taste modifier made of curculin can be processed into powder, solution, sheet, spray, emulsion, etc. depending on the properties of the food.

[Effect]

The curculin of the present invention acts on the receptor membrane of the tongue when eating sour substances, tasteless substances, water, etc., or when eaten together with sour substances, tasteless substances, water, etc. after being ingested during the day. It has a deceptive effect, and if you eat the curculin of the present invention or a preparation or food containing the curculin in advance, you will not notice the sweet taste when you eat sour substances, tasteless substances, or water until 30 minutes later. It can make you feel.

Pure Curculin (Curculin 4X10) for various aqueous solutions
-'M concentration) is as shown in Table 2 below, and the activity is particularly high against sour substances.

In addition, the curculin of the present invention is stable even in an aqueous solution, unlike the miraculin contained in the fruit of the miracle fruit. It is easy to make it into physical form or spray form.

Moreover, the curculin of the present invention itself has a weak sweet taste.

〔Example〕

Next, examples will be shown to specifically explain the present invention.

Example 1 Curculigo latifolia (Curculigo Ia)
tif.

Add 600 m of water to 30 g of freeze-dried fruit bulb of 1 ia), homogenize for 2 minutes with a mixer, and then
Centrifuge for 30 min at 000 r, p, m, and remove the supernatant (
After removing the residual fragrance, 600 mfl of water was added to the remaining fragrance, and homogenization, centrifugation, and removal of the supernatant were repeated four times until the supernatant was no longer colored, to obtain the residual fragrance.

Next, 250 d of a 0.5 M NaCI aqueous solution was added to this residual aroma, homogenized for 2 minutes with a mixer, and then filtered with suction. After separating the filtrate, 250 d of 0.5M NaCl aqueous solution was added to the residual aroma, and homogenization and suction filtration were performed.
The filtrate was separated.

Next, after combining the filtrates, 1 at 30000 r, p, m.
The mixture was centrifuged for hours to obtain a supernatant (crude curculin extract).

This crude extract was desalted and freeze-dried to obtain a curculin-containing substance award (crude luculin).

Example 2 After concentrating 500 ml of the crude curculin extract obtained in Example 1 to 30 d by ultrafiltration, this concentrated solution was
．． 01M phosphate buffer (pl+6.8) 10mft
was added to make the sample solution 100 rtdl.
CM-Sepharose column chromatography was performed (CM-Sepharose CL-6B, flattened with 0.01M phosphate buffer (pl+6.8)).Bed volume: 130mff1 and bend height: 17c.
The sample solution was poured into the m0 column, washed with 0.01M phosphate buffer (pH 6, 8), and then 0-1, OMNaC
I/0.01 M phosphate buffer y-(pH 6, 8)
It was eluted at the end of the gradient, and the active fractions were collected.

This active fraction was desalted and lyophilized to obtain a curculin-containing substance (crudely purified curculin).

Example 3 After concentrating the active fraction obtained in Example 2 by ultrafiltration,
Gel column (Toyo Soda TSK gel G3000SW)
The mixture was subjected to HPLC using 0.01M phosphate buffer (pH 6,8) and eluted with 0.01M phosphate buffer (pH 6,8).

Curculin was eluted as one sharp peak with strong activity.

This active fraction was desalted and lyophilized to obtain purified curculin.

In addition, this purified curculin was dissolved in 5.0 M urea in the presence of 8.0 M urea.
When subjected to DS-PAGE, the molecule ff112500
A single band was observed at the Dalton region, confirming that it was a pure curculin product.

Curculigo latifolia fruit freeze-drying bulb 30
The protein amount of curculin obtained in each example per g,
The activity yield and specific activity were as shown in Table 3 below.

Example 4 10 mg of crude curculin obtained by t% in Example 1 and 1 m of common salt
g was dissolved in 20 ml of water to make a 0.05% aqueous solution.

This aqueous solution remained stable even after being left at room temperature (around 25°C) for one month. After injecting 1 aft of this aqueous solution for 1 minute during the day and spitting it out, the subjects ate and drank 0.02 M citric acid, water, and black tea, respectively. The perceived sweetness at that time was as shown in Table 4 below.

In the case of black tea (black) in Table 4, the taste was similar to that of regular black tea with added sugar.

Example 5 Black coffee jelly (A) and sugar-containing coffee jelly (B) were prepared using the formulations shown in Table 5 below.

Table 5 On the other hand, 0.005% of the coarse luculin obtained in Example 1 was added to commercially available fresh cream (C) to prepare curculin-containing fresh cream (D).

After dissolving the coffee jelly (A), pour it into a cup, cool it and solidify it into a jelly, then squeeze about 10g of whipped cream (D) with 10% sugar added to it.
Got cream coffee jelly. In addition, cream coffee jelly made by whipping fresh cream (C) with 10% sugar added on top of coffee jelly (B),
Obtained in the same manner as above.

After eating whipped cream (D), coffee jelly (A)
), the taste was similar to that of eating coffee jelly (B) after eating whipped cream (C).

Example 6 Instead of the whipped cream (D) used in Example 5, 0.2 g of an edible film made by adding 0.2% of the crude curculin obtained in Example 1 was added to prevent drying. A thin film was placed on the surface of the coffee jelly (A) in No. 5. When I ate the coffee jelly (A) after eating this edible film, I felt the same flavor as when I ate the coffee jelly (B) of Example 5.

Example 7 Curculigo latifolia (Curculigo 1a)
tif.

Add 600 ml of water to 30 g of dried fruits of 1ia), homogenize for 2 minutes with a mixer, and then
Centrifuged for 30 minutes at r,p,m. After removing the supernatant (colored), add 600 mft of water to the remaining liquid,
Homogenization, centrifugation, and supernatant removal were repeated 4 times until the supernatant was no longer colored, and the remainder was obtained.

Next, add 250 y of 0.5 M NaCl aqueous solution to this balance.
After adding n'l and homogenizing with a mixer for 2 minutes,
Filtered with suction. After separating the filtrate, add 0.5 MNa to the remaining balance.
C1 aqueous solution 250#11! was added, homogenized and suction filtrated, and the iIt liquid was fractionated.

Next, after combining the filtrates, 30000 r, p, +a,
The mixture was centrifuged for 1 hour to obtain a supernatant (crude curculin extract).

This crude extract was desalted and dried to obtain a curculin-containing substance (crude luculin).

Example 8 After concentrating 500 μm of the crude curculin extract obtained in Example 7 to 30 m by ultrafiltration, 0.
CM-Sepharose column chromatography was performed using the sample solution prepared by adding 70 d of 0.01 M phosphate buffer (pH 6,8") to 100 d (CM-Sepharose CL-6B, 0.01 M phosphate buffer (pH 6,8").
6, 8), ], bed volume 13
The sample solution was poured into a column with a bed height of 17 CTl and a bed height of 17 CTl, washed with 0.01 M phosphate buffer (pH 6, +3), and then washed with 0-1, 0 M NaCl/0. OIM
It was eluted with a gradient end of phosphate buffer (pH 6, 8) and the active fraction was collected.

This active fraction was desalted and dried to obtain a curculin-containing substance (crudely purified curculin).

Example 9 After concentrating the active fraction obtained in Example 8 by ultrafiltration,
Gel column (Toyo Soda TSK gel G3000 SW
) and subjected to HPLC using 0. Elution was performed with OIM phosphate buffer (pH 6,8).

Curculin was eluted as one sharp peak with strong activity.

This active fraction was desalted and dried to obtain purified curculin.

When this purified curculin was subjected to 5DS-PAGE in the presence of 8.0 M urea, a single band was observed at a molecular weight of 12,500 daltons, confirming that it was a pure curculin product.

The protein content, activity yield, and specific activity of curculin obtained in each example per 30 g of dried fruit of Curculigo latifolia were as shown in Table 6 below.

Example 10 10011 g of crude talcrine obtained in Example 7 and 1 salt
mg was dissolved in 20 ml of water to make a 0.5% aqueous solution.

This aqueous solution remained stable even after being left at room temperature (around 25°C) for one month. After holding 1 ml of this aqueous solution in the mouth for 1 minute and spitting it out, the subjects drank and drank 0.02 M citric acid, water, and black tea, respectively. At that time, the taste was as shown in Table 7 below.

It tasted similar to tea.

Example 11 Black coffee jelly (A) and sugar-containing coffee jelly (B) were prepared using the formulations shown in Table 8 below.

On the other hand, 0.05% of the crushed luculin obtained in Example 7 was added to commercially available fresh cream (C) to prepare curculin-containing fresh cream (D).

After dissolving the coffee jelly (A), pour it into a cup, cool it and solidify it into a jelly, then squeeze about 10g of whipped cream (D) with 10% sugar added to it.
I got cream coffee jelly. Also, sand IJ on top of coffee jelly (B) and fresh cream (C)! Cream coffee jelly topped with 10% whipped material was obtained in the same manner as above.

After eating whipped cream (D), coffee jelly (A)
), I felt the same flavor as when I ate coffee jelly (B) after eating whipped cream (C).

Example 12 Instead of whipped cream (D) used in Example 11,
0.2 g of an edible film prepared by adding 0.5% of the coarse lucrin obtained in Example 7 was placed in the form of a ril film on the surface of the coffee jelly (A) of Example 1 to prevent drying. After eating this edible filler 1, the coffee jelly (A) was eaten.
The flavor was similar to that when eating B).

Examples 13 to 18 Instead of the 0.5M NaCl aqueous solution used in Example 7, 0.
．． 5M KCl aqueous solution (Example 13), 0.5M CaCI
z aqueous solution (Example 14), 0.5M Na1lCO8 aqueous solution (Example 15L, 0.5MMgC0, aqueous solution (Example 16), 0.2MKH,PO, aqueous solution (Example 1)
7), 0.2M N a HtP O-aqueous solution (
Curculin-containing substances (crude curculin) were obtained in the same manner as in Example 7, except that Example L8) was used.

[Effects of the Invention] The novel substance curculin of the present invention has a high degree of stabilization and is suitably used as a taste modifier. Sweetness that is desirable when eating foods that have to be acidic for reasons such as preservation, or foods that do not contain sugar for health and nutritional reasons. It can be used for a wide range of purposes because it can confuse people.

Claims (5)

[Claims] (1) Protein curculin obtained by extracting the fruit of Curculigo latifolia or its dried product with an aqueous salt solution having a concentration of 0.01M or more. (2) Taste modifier consisting of curculin. (3) Food containing a taste modifier consisting of curculin,
drinks or drugs. (4) The food, drink, or drug according to claim (3), wherein the food, drink, or drug is processed into a powder, solution, sheet, tablet, spray, or emulsion. (5) A method for extracting the protein curculin, which comprises extracting the fruit of Curculigo latifolia or its dried product with an aqueous salt solution having a concentration of 0.01M or more.