JP2993724B2 - Stabilized yellow dye - Google Patents

Description

The present invention relates to a natural yellow pigment, a method for producing the same, and uses thereof.

[Conventional technology]

Glycosides of carotenoid pigments, especially gardenia yellow pigments, are inferior in light resistance and acid resistance, and their use range is limited. For example, it has been pointed out that crocin loses gentiobiose, which is a saccharide thereof, due to the action of an enzyme β-glucosidase, an acid, or an alkali contained in foods and beverages to be used, becomes crocetin, and significantly reduces the solubility in water. I was

For the purpose of improving the stability of the dye, a method of adding cyclodextrin (JP-A-54-117536), a method of adding ascorbic acid and polyphosphoric acid (JP-B-51-37336).
JP-A-62-1269), and a method of adding an extract of teas
No. 53) is known.

However, in these methods, although a certain improvement in stability is observed, the improvement is not enough to endure practical use, or because other than the original dye component is added, the action of the additive causes a decrease in solubility, for example. It is pointed out that difficulties such as enhancement of acidity or bitterness and color tone change may occur. Furthermore, the above method has no effect of inhibiting the decomposition into crocetin. In addition, as a method for improving the stability of a pigment having a glycoside, there is also known a method in which a sugar such as cyclodextrin or dextrin is subjected to a transfer reaction using an enzyme cyclodextrin glucanotransferase in a glycoside of an anthocyanin-based pigment. (Japanese Unexamined Patent Publication (Kokai) No. 63-43959), a glycosyl transfer dye to a glycoside of a dye other than anthocyanin has not yet been known.

[Problems to be solved by the invention]

The present invention provides a novel carotenoid pigment, particularly a pigment obtained by transfer-condensation of various sugars using a transferase to a yellow pigment containing crocin as a main component obtained from gardenia fruits, and the problems involved in the prior art are provided. In particular, it is intended to improve the stability of the dye.

[Means for solving the problem]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, various sugars, such as starch, glycogen, and the like, in a yellow pigment mainly containing crocin obtained from gardenia fruits.
Cyclodextrin, dextrin, and other oligosaccharides, for example, lactose, sugar, xylo-oligosaccharides, etc., alone or in combination of two or more thereof, with crosin as the main component in the presence of the corresponding transferase By treating the yellow pigment, a novel pigment in which various carbohydrates are condensed can be obtained, and this pigment has better heat stability at a lower pH than conventional known gardenia yellow pigment, and is stable to light. In addition, the inventors have found that the stability to β-glucosidase is increased, and completed the present invention.

Therefore, according to the present invention, there is provided a novel carotenoid pigment in which a carbohydrate is condensed to a hydroxyl group of a glycoside of a carotenoid pigment, and a method for producing the carotenoid pigment and a use thereof are provided.

 Hereinafter, the present invention will be described specifically.

The carotenoid pigment of the present invention includes a wide variety of carbohydrate condensates depending on the type of the starting material pigment used for the production and the kind of carbohydrate donating a new sugar chain (referred to as “donor carbohydrate”). Is done. Such a carotenoid pigment may be of any origin as long as it can be produced by utilizing the enzymatic transfer reaction of the present invention as described in detail later, but particularly the gardenia yellow pigment, that is, the glycoside of crocin 1-a and FIG. 2, FIG. 4 and FIG. 5, FIG. 6 and FIG. 7, and FIG. Dye mixtures as specified by FIG. 9 as well as their constituent dyes can be mentioned. The term “condensed” as used in the present invention refers to a product obtained by dehydrating and condensing a hydroxyl group of a donor saccharide by a corresponding glycosyltransferase to a hydroxyl group of a sugar chain (derived from gentiobiose) of crocin. This condensation differs depending on the enzyme used. In the case of cyclodextrin glucanotransferase, 1 to 6 (mainly 6) glucoses are bonded to the hydroxyl group at the 4-position of the sugar chain via α-1,4 bonds. However, in the case of α-glucosidase, glucose is α-1,6 bonded to the hydroxyl group at position 6 of the sugar chain, and in the case of β-galactosidase, galactose is β-1 ,, at the hydroxyl group at position 4 of the sugar chain. It is presumed that the one with four bonds is the main. Therefore, the dyes of the present invention provide crocin dyes in which various sugar chains are condensed depending on the donor saccharide, transferase and / or reaction time used.

These pigments are more specifically another invention of the present invention, in which a glycosyltransferase of the carotenoid pigment and a glycosyltransferase are used to cause a transfer reaction in the presence of a carbohydrate to thereby form the glycoside. It can be produced by a method characterized by condensing the saccharide with a hydroxyl group. Since various types of glycosyltransferases used in the present invention have an appropriate combination depending on the donor saccharide used, it is preferable to select and use the type of enzyme according to the saccharide to be condensed. That is, when saccharide such as dextrin, starch or maltooligosaccharide is condensed with crocin, a gardenia yellow pigment, cyclodextrin-glucanotransferase (CG
Tase EC2.4.1.19), preferably C from Bacillus sp.
The dyes of the present invention can be produced by allowing GTase to act. Similarly, lactose as a saccharide, β-galactosidase when using galactooligosaccharides, invertase or sucralase when using sugar, xylan, and xylanase when using xylo-oligosaccharides act on the xylanase of the present invention. Pigments can be produced. Depending on the enzyme used, CG
In the reaction of Tase, yellow pigment mainly composed of crocin
1-8% (preferably 0.5-4%), dextrin 0.5-4
0% (preferably 5 to 20%) CGTase 5 units / g dextrin or more (preferably 10 units / g dextrin or more)
Is used. The buffer used and its concentration can be freely selected as long as they do not inhibit the enzyme reaction. For example, a 50 mM phosphate buffer may be mentioned. Its pH is 5-8
(Preferably 6) is appropriate, the reaction temperature is suitably 35 to 50 ° C., and the composition of the reaction mixture may be appropriately confirmed by liquid chromatography to terminate the reaction.

The reaction product may be separated and purified by a conventional method, that is, by using an adsorption resin or ultrafiltration or another method.The purified dye solution may be concentrated or freeze-dried to obtain the stabilized yellow dye of the present invention. Can be obtained individually or as a mixture.

Upon use, the solution obtained by the above method may be used as it is, or may be used after being appropriately diluted or concentrated, or may be dried and used in a powdered state if desired. Further, they can be used as they are or in combination with other dyes depending on the purpose of use.

〔Example〕

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

Example 1 Gardenia yellow pigment (color value ▲ E ^10W% _1cm ▼ = 2000) 0.5
g and 5 g of dextrin are dissolved in 100 mM of 50 mM phosphate buffer (pH 6), and the enzyme CGTase (Antano Pharmaceutical Co., Ltd. Contizyme) is dissolved.
25 units were added and reacted at 45 ° C. for 24 hours. The obtained dye mixture of the present invention shows the composition shown in the chromatogram in FIG. 1 and the absorption spectrum in the ultraviolet and visible regions in FIG.

Color Tone Stability This dye and a yellow dye mainly composed of crocin as a control were prepared in McClubane buffer at pH 3 or 6, respectively.
Absorbance after irradiation for 20 hours, 1 hour at 100 ° C, 4 days at 37 ° C,
Table 1 shows the absorbance after holding for 7 days.

From the above results, this dye has a particularly low pH than the raw material dye itself.
It was found to show significant stability in the region.

Enzyme stability β-glucosidase (Sumizyme AC) was added to the present dye at 83 units / g, and reacted at 50 ° C for 2 hours. The HPLC chromatograms before and after the reaction were respectively expressed as 3-a
This is shown in the figure and FIG. 3-b. Similarly, a yellow pigment mainly containing crocin as a raw material is similarly treated with β-glucosidase,
The HPLC chromatograms before and after the reaction are shown in FIGS. 3-c and 3-d, respectively. As described above, the present dye is hardly decomposed into crocin and crocetin even by the above-mentioned enzyme treatment, and exhibits excellent enzyme stability.

Example 2 Gardenia yellow pigment (color value ▲ E ^10W% _1cm ▼ = 2000) 0.01
g and lactose (0.5 g) were dissolved in 10 mM 100 mM phosphate buffer (pH 7.2), and β-galactosidase (0.75 units) was added, followed by reaction at 50 ° C. for 6 hours. The obtained pigment mixture of the present invention is
4 shows the composition shown in the chromatogram in FIG. 4 and the absorption spectrum in the ultraviolet and visible regions in FIG.

Color Tone Stability The absorbance of this dye at pH 3 or 6 in McClubane buffer at the time of preparation and after irradiation for 20 hours at 12,000 lux fluorescent lamp, after 1 hour at 100 ° C, 4 days at 37 ° C and 7 days The absorbance is shown in Table 2.

Example 3 Gardenia yellow pigment (color value ▲ E ^10W% _1cm ▼ = 2000) 0.01
g, dextrin 0.5g, 50mM phosphate buffer (pH6) 10m
And add 10 units of α-glucosidase,
For 2 hours. The obtained dye mixture of the present invention has the composition shown in the chromatogram in FIG. 6 and the absorption spectrum in the ultraviolet and visible regions in FIG.

Color tone stability The absorbance after preparation with McClubane buffer of pH 3 or 6 and after irradiation with 12,000 lux fluorescent lamp for 20 hours, and after holding at 100 ° C for 1 hour, 37 ° C for 4 days and 7 days, were measured as the third values. It is shown in the table.

The chromatogram for the stabilized yellow dye of the present invention was obtained by high pressure liquid chromatography (HPLC) (YMC ODS A-312 manufactured by Yamamura Chemical Laboratory, diameter: 0.6 cm, length: 15 cm, solvent:
50% methanol, 1m / min, detection: spectrophotometer, 440n
m, Recorder: The results of analysis with a Shimadzu CR-3A) are shown.

Example 4 Purification of Stabilized Yellow Dye-1 The dye obtained in Example 1 was adsorbed on an adsorption resin (Diaion HP
-20 (manufactured by Mitsubishi Kasei), washed thoroughly with water, and then eluted the dye with an 80% methanol aqueous solution.
85 g of stabilized yellow pigment powder were obtained.

Example 5 Purification of stabilized yellow pigment-2 0.1 g of the stabilized yellow pigment powder obtained in Example 4 was subjected to gel filtration resin (Sephadex LH-20 Pharmacia) 2.8c.
When loaded onto a column of mφ × 140 cm and eluted with 50% acetone, the sugar-modified (condensed) dye eluted between 190 and 300 m. The chromatogram is shown in FIG.

Example 6 The sugar modification of crocin by the fermentation method was performed by the following method. That is, Bacillus maceran
s) (IF03490) crocin 0.1%, corn steep liquor 1.0%, soluble starch 5.0%, ammonium sulfate 0.5
% And calcium carbonate at 1.0%, and cultured at 30 ° C. for 3 days to modify crocin with sugar. On the 3rd day of culture
The chromatogram by HLPC is shown in FIG.

Chinese noodles were produced according to a conventional method using the above blends.

(Results) The color tone of this example containing crocin glycoside was
The flavor, heat resistance and light resistance were superior to those of the comparative example.

Using the above composition, 50 L of syrup was prepared according to a conventional method. 50 m of the syrup was diluted with carbonated water and filled into a bottle to make a total volume of 200 m.

(Results) This example was superior to the comparative example in light resistance, heat resistance, color tone and flavor, and did not show browning.

〔The invention's effect〕

As shown in Example 1 above, the yellow dye of the present invention produced by the above method has better heat and light stability at a low pH than the above-mentioned conventional yellow dye, and is more stable to hydrolysis by enzymes. The nature is also high. Therefore, liquor,
It is also excellent as a coloring agent for processed protein products such as foods, beverages, cosmetics, pharmaceuticals, noodles, and kamaboko.

[Brief description of the drawings]

FIG. 1-a, FIG. 4, FIG. 6 and FIG. 9 are chromatograms by HPLC of the dye mixtures produced in Examples 1, 2, 3 and 6, respectively, and FIG. 1-b. Represents the same chromatogram of the raw gardenia yellow pigment, FIGS. 2, 5 and 7 show the absorption spectra of the above mixture in the ultraviolet and visible parts, respectively. FIG. Before the β-glucosidase treatment of the stabilized dye obtained in the above, FIG. 3-b shows the HPLC chromatogram after the treatment, FIG. 3-c shows the HPLC chromatogram before the treatment of the raw material dye, and FIG. FIG. 8 shows a chromatogram of the mixture of Example 2 after gel filtration chromatography. The units on the X-axis in FIGS. 1-a, 1-b and 3 and the units on the X-axis in FIGS. 4, 6 and 9 respectively represent the retention time (minutes), and The units on the X-axis in FIGS. 2, 5 and 7 each represent wavelength (nm), and the units on the Y-axis each represent absorbance.

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C09B 61/00 C12P 19/18 A23L 1/275

Claims (4)

(57) [Claims] 1. A novel carotenoid pigment in which a saccharide is condensed with a hydroxyl group of a glycoside of a carotenoid pigment. 2. The method according to claim 1, wherein the carbohydrate is condensed with a hydroxyl group of the glycoside by causing a transfer reaction using a glycosyltransferase in the presence of the carotenoid pigment glycoside and carbohydrate. Item 10. A method for producing the dye according to Item 1. 3. A food comprising the pigment according to claim 1. 4. A coloring agent comprising the coloring matter according to claim 1.