JP3398463B2 - Stabilization method for foods containing coloring agents and / or vitamins - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for stabilizing coloring agents and / or vitamins.

[0002]

2. Description of the Related Art Flavonoids such as hesperidin, rutin, and naringin have been known to stabilize coloring agents and vitamins due to their strong ultraviolet absorption in addition to their medicinal effects. ing. However, since these flavonoids are hardly soluble in water, the amount that can be added to the target is limited. The inventors of the present application have glycosylated these flavonoids to efficiently produce solubilized flavonoids (Japanese Patent Application No. 5-25614).
No. 1) was developed.

[0003] Among these solubilized flavonoids, α-
Since glucosyl rutin and α-glucosyl quercetin are yellow, their color tone changes when added to an object. Therefore, the amount that can be added to the object is limited.

[0004]

Means for Solving the Problems The solubilized hesperidin used in the present invention is mainly a compound in which glucose is bound at the 4-position of glucose of hesperidin by an α-1,4 bond (hereinafter, this compound is referred to as “α-glucose”). Pyranosyl hesperidin)) and glucose in the 4-position of the glucose, further comprising one to ten or more α-1,4 bonds.

[0005] The solubilized naringin used in the present invention is such that glucose is α-1,
A compound bonded by four bonds (hereinafter, this compound is referred to as “α-glucopyranosylnaringin”) and glucose is further added to the 4-position of glucose by α-1,4 bonds.
It is a mixture mainly consisting of a dozen or more bonded.

[0006] The object of the present invention has a colorant (preferably 0.01 to 1% by weight based on the object) and / or a vitamin (preferably 0.01 to 1% by weight based on the object) dissolved therein. Liquid, gel or solid foods and drinks, cosmetics, pharmaceuticals, etc. (hereinafter collectively referred to as foods, etc.).

Colorants include crocin (a carotenoid pigment), phycocyanin (a porphyrin pigment), beet red (a betacyanine pigment), safflower yellow pigment (a flavonoid pigment), anato pigment (a carotenoid pigment) and cochineal (anthraquinone pigment). ) And the like are not particularly limited, but those with poor stability are targeted.

The vitamin is a normal vitamin, for example, vitamins A, B, C and D.

The present invention is particularly effective when the object is contained in a transparent or translucent container and is exposed to sunlight or fluorescent light. Usually, 0.01 to 1% by weight of the solubilized hesperidin or solubilized naringin is sufficient from the viewpoint of effectiveness. The solubilized hesperidin or solubilized naringin is made into a solution having a high concentration and mixed or dispersed in the object.

[0010] Solubilized hesperidin or solubilized naringin is CGTavse, ie, 1,4-α-D-gluca.
n; 4-α-D- (1,4-glucano) -tra
nsferase (EC 2.4.1.19.) can be used for production (Patent Application 5-25).
No. 6141). Any enzyme may be used for production as long as it is CGTase. However, the use of a substance which is unstable in an alkaline region significantly reduces the yield of solubilized hesperidin or solubilized naringin.

A strain B2-5a of the genus Bacillus developed by the inventors of the present application (Funded Deposit F of the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology)
A novel CGTase collected from a culture of ERM P-13864 (hereinafter referred to as the present strain) is preferably used because it has high activity in an alkaline region. The culture conditions of this strain and the method of collecting the enzyme are not exceptional.

The conditions for the transglycosylation to hesperidin or naringin using the novel CGTase are not special, as in conventional methods, except that they are performed in an alkaline region. Preferably pH 8-11, temperature 20-
75 ° C., donor concentration 0.1-30%, preferably 1-30%
20%, the concentration of hesperidin or 0.1 to 5%, preferably 0.5 to 2%, and the enzyme is 0.1 to 100 units /
The reaction is started to make up to ml. The longer the reaction time, the better the enzyme activity is. The reaction is 100
Stop by heating at 5 ° C. for 5 minutes.

As the donor, α-1,4-glucans having α-1,4 bonds, such as cyclodextrin and maltooligosaccharides, various kinds of oligosaccharides, and starch are used.

As an example of the above procedure, solubilized hesperidin was produced as follows and then isolated and its structure determined. 0.5% hesperidin, 5% soluble starch, 1,
Reaction solution 600m containing 200 units of CGTase
were reacted at 40 ° C. for 16 hours. After the reaction, the reaction was stopped by heating at 100 ° C. for 5 minutes. To this, 10 units of β-amylase were added and reacted at 40 ° C. for 1 hour.
The reaction was stopped by heating at 00 ° C. for 5 minutes. This was applied to an Amberlite XAD-16 column, and the column was washed with distilled water.
Hesperidin glycoside adsorbed on No. 16 was eluted. The eluate was concentrated and purified by preparative FPLC (column: ODS, solvent: 20% ethanol, flow rate: 2 ml / min). Further, this was used for preparation TLC (silica gel plate, developing solvent; chloroform: methanol: distilled water =
65:35:10 upper layer, developed by the ascending method), scraping spots having an absorption at 253 nm from the silica gel plate, extracting with distilled water, and lyophilizing them.
Compounds which are considered to be hesperidin monoglucoside and diglucoside as main reaction products are each 4.2.
mg, 15.4 mg.

The compounds which are considered to be hesperidin monoglucoside and diglucoside purified as described above are 10 units / ml of glucoamylase or α-
Treatment with glucosidase at 40 ° C. for 16 hours produced hesperidin and glucose in a molar ratio of about 1: 1 and 1: 2, respectively. In addition, it was not decomposed by β-glucosidase treatment under the same reaction conditions. This indicates that these compounds contain hesperidin in the form of α at a molar ratio of glucose of 1: 1 and 1: 2, respectively.
It was found that hesperidin monoglucoside and diglucoside linked at −1,4.

The hesperidin released by this enzyme treatment was analyzed by HPLC (ODS) described below, and the released glucose was analyzed by the glucose oxidase method (I. Mi.
waetal. Clin. Chem. Acta , 37,
538-540 (1972)). HPLC (ODS) analysis conditions

Further, the structure of hesperidin monoglucoside was analyzed in detail by methylation analysis. Dissolve fully dried hesperidin monoglucoside in 0.25 ml of dimethyl sulfoxide, add freshly prepared methylsulfinyl carbanion,
The reaction was performed at room temperature for 2 hours. To this, 0.15 ml of methyl iodide was added and reacted at room temperature for 4 hours to carry out methylation. The methylated product was placed in 90% (v / v) formic acid at 100 ° C. for 1 hour.
Then, the mixture was hydrolyzed in 1N sulfuric acid at 200 ° C. for 4 hours.
Add 13 mg of NaBH ₄ to the methylated product and add
After reducing for an hour, the mixture was acetylated by reacting at 100 ° C. for 1 hour in 0.5 ml of an acetic anhydride / pyridine mixture (1: 1, v / v). The partially methylated alditol acetate thus prepared was analyzed by gas chromatography (GC). The analysis conditions of GC are as follows. Hesperidin and maltosyl- were used as standard samples.
β-cyclodextrin was used.

[0018]

[Table 1]

The results of the analysis by GC are shown in Table 1. 2,3,4,6-te from hesperidin monoglucoside
tra-O-methyl-glucitol ace
state, 2,3-di-O-methyl-gluc
itol acetate, 2,3,6-tri-O-
Since methyl-rhamnitol acetate was detected, the glucose transferred by CGTase was α-positioned at the 4-position of hesperidin glucose.
It was revealed that they were bonded by 1,4 bonds, that is, α-glucopyranosyl hesperidin.

FIG. 2 shows a HPLC (ODS) chromatograph (analysis conditions are as described above) when the hesperidin glycoside was treated with β-amylase.
From this, it is considered that a series of hesperidin glycosides has sugar bound to hesperidin in maltose units.

From the results of these enzyme treatments and methylation analysis, the solubilized hesperidin was mainly composed of α-glucopyranosyl hesperidin and the glucose at the 4-position of glucose, and several to ten to ten or more α-1,4 bonds. It turned out to be a mixture consisting of singly bonded ones.

Similarly, α-glucopyranosyl naringin can be isolated from solubilized naringin.

[0023]

The absorption spectrum of the reaction product obtained by the above-mentioned production method, that is, the solubilized hesperidin, is examined.
The absorption spectrum was compared with that of a control solubilized rutin (FIG. 1). As shown in FIG. 1, the solubilized hesperidin has a strong ultraviolet absorption. 350-400nm
It was found that there was no absorption in the vicinity, and it was extremely colorless.
On the other hand, solubilized rutin showed strong absorption at that wavelength. That is, it exhibits a deep yellow color. The solubility of α-glucosyl hesperidin (monoglucoside) or diglucoside in water was about 350 or 420 times higher than that of hesperidin. The molar extinction coefficients of α-glucosyl hesperidin (monoglucoside) and diglucoside were 8900 and 6800, respectively.

Comparative Example 1 Solubilized hesperidin was added to a solution containing 0.05% (% by weight) of phycocyanin (a porphyrin dye) at 0.01%, 0.05%, and 0.1% (% by weight). Put into test tubes, test plots 1, 2, 3
And At the same time, 0.01%, 0.05%, 0.1%
% (% By weight) solubilized rutin was added to test groups 4, 5, and 6, respectively. Leave them at 4 ° C,
Irradiate from four sides with a fluorescent lamp of 000 lux, measure absorbance at 620 nm of maximum absorption of phycocyanin with time, and absorbance at the start of standing (0.6 in this case)
Was set to 100, and the amount of remaining dye was shown as a percentage. At this time, a phycocyanin solution containing no solubilized hesperidin and solubilized rutin was used as a control. The control group gradually faded (absorbance decreased) over time,
After 0 hour and 100 hours, the remaining amount of the dye is 3
0% and 20%. On the other hand, in the test plot, the dye was stabilized, and the amount of the residual dye after 50 hours was less than that in test plots 1, 2, and 3.
Were 65%, 75% and 80%, respectively. In Test Zones 2 and 3, the original color tone was visually maintained. After 100 hours, they were 45%, 55% and 60%, respectively. On the other hand, the test plot to which solubilized rutin was added, especially
In the test plots 5 and 6, the blue color of phycocyanin turned green because the solubilized rutin had a yellow color tone.

[0025]

【Example】

(Example 1) 0.01%, 0.05%, 0.1% (% by weight) of solubilized hesperidin was added to a solution containing 0.05% (% by weight) of crocin (a carotenoid type pigment), a gardenia pigment. , Put in test tubes and test sections 1, 2, 3 respectively
And These are allowed to stand at 4 ° C., and 12,000 lux
And the absorbance at 442 nm of the maximum absorption of crocin was measured over time, and the absorbance at the start of standing was defined as 100, and the residual amount of the dye was shown as a percentage. At this time, a crocin solution containing no solubilized hesperidin was used as a control. The control section gradually faded (absorbance decreased) over time, and for 4 hours,
After the elapse of 8 hours, the amount of the remaining dye was 25% and 5%, respectively. On the other hand, the dye was stabilized in the test plot, and the amount of the residual dye after 4 hours was 45
%, 60% and 75%. In Test Zone 3, the original color tone was visually maintained. After 8 hours, they were 10%, 25% and 50%, respectively.

Example 2 0.005% (% by weight) of riboflavin which is vitamin B ₂ and a yellow pigment
0.01%, 0.05% solubilized hesperidin
% And 0.1% (% by weight) were added and placed in test tubes to make test groups 1, 2, and 3, respectively. Leave them at 4 ° C.
Irradiation was carried out from all sides with a 2,000 lux fluorescent lamp, the absorbance was measured over time at a maximum absorption of riboflavin of 445 nm, and the absorbance at the start of standing was defined as 100, and the remaining amount of riboflavin was shown as a percentage. At this time, a riboflavin solution containing no solubilized hesperidin was used as a control. The control group rapidly faded (absorbance decreased) with the passage of time, and after 2 hours, the remaining amount was 10
%. In comparison, riboflavin in the test plots was stabilized, and the residual amount after 2 hours was 45%, 60%, and 70% in test plots 1, 2, and 3, respectively. Further, even after 4 hours, the residual amount in the test section 3 was 50% or more.

Example 3 Solubilized hesperidin was added to a solution containing 0.1% (% by weight) of cochineal (anthraquinone dye) in an amount of 0.01%, 0.05%, and 0.1% (% by weight). Test tubes 1, 2, and 3 were placed in test tubes, respectively. These were allowed to stand at 4 ° C., illuminated from all directions with a 12,000 lux fluorescent lamp, and the absorbance was measured over time at 525 nm of the maximum absorption of cochineal. Was shown as a percentage. At this time, a cochineal solution containing no solubilized hesperidin was used as a control. The control group gradually faded (absorbance decreased) with the passage of time.
After the elapse of time, the amount of the remaining dye was 20% and 10%, respectively. On the other hand, the test plot showed stabilized dye and 10
The residual amount of the dye after 25 hours was 25 in each of the test groups 1, 2, and 3.
%, 50% and 75%. Further, the residual amount of the dye in Test Section 3 was still 75% after 48 hours. In Test Section 3, the original color tone was visually maintained.

Example 4 α-Glucopyranosyl hesperidin was added to a solution containing 0.05% (% by weight) of crocin (a carotenoid pigment) as a gardenia pigment.
%, 0.05%, and 0.1% (% by weight) were added to test tubes to prepare test groups 1, 2, and 3, respectively. These were allowed to stand still at 4 ° C., illuminated from all sides with a 12,000 lux fluorescent lamp, and the absorbance was measured with time at the maximum absorption of crosin of 442 nm. Was shown as a percentage. At this time, a crocin solution containing no solubilized hesperidin was used as a control. As a result, the initial color tone of α-glucopyranosyl hesperidin was visually maintained in Test Groups 2 and 3.

Example 5 A juice containing 0.05% (% by weight) of a red cabbage dye (containing anthocyan) and a solubilized hesperidin in a juice having the composition shown in Table 2 was added in an amount of 0.01% and 0.1%.
05% and 0.1% (% by weight) were added, and the juice was prepared by the usual production method.
And These were allowed to stand at 4 ° C., and irradiated from all sides with a 12,000 lux fluorescent lamp, and the maximum absorption of anthocyan at 538 nm was measured over time. At this time, a juice containing no solubilized hesperidin was used as a control. The control group gradually faded (absorbance decreased) with the passage of time, and the residual amount of the dye became 50% after 24 hours. In contrast, the dyes were stabilized in the test plots, and the residual dye amount after 24 hours was 75% or more in all the test plots. These were visually judged to have maintained the original color tone and had sufficient commercial value. Further, in the test section 3, the color tone was stable even after 24 hours, and the amount of the remaining dye did not decrease. In addition, the taste and color tone of the juice were not changed by the solubilized hesperidin at the above addition amount.

[Table 2]

Example 6 Phycocyanin was 0.1%
(% By weight) 0.01%, 0.05%, 0.1% of solubilized hesperidin in ice cream having the formulation shown in Table 3 containing
(% By weight), ice cream was prepared by a usual production method, and placed in a transparent plastic container (without a lid) to obtain test groups 1, 2, and 3, respectively. These were allowed to stand at −20 ° C., irradiated with a 12,000 lux fluorescent lamp from all sides, dissolved over time, and the maximum absorption of phycocyanin was measured at 620 nm. At this time, an ice cream containing no solubilized hesperidin was used as a control. The control group gradually faded (absorbance decreased) with the passage of time, and after 48 hours, the residual amount of the dye became 35%. On the other hand, the dye was stabilized in the test plot, and the amount of the dye remaining after 48 hours was in the test plot 1,
It was 70%, 75%, and 85% for 2, 3, respectively. In particular, in Test Zones 2 and 3, the initial color tone was visually maintained, and it was judged that the samples had sufficient commercial value. In addition, the taste and color tone of the ice cream were not changed by the solubilized hesperidin at the above addition amount.

[Table 3]

Example 7 The same test as in Example 1 was conducted for solubilized naringin. As a result, similar results were obtained with solubilized hesperidin.

Example 8 The same test as in Example 4 was conducted for α-glucopyranosylnaringin. As a result, similar results were obtained with α-glucopyranosyl hesperidin.

[0033]

[Effect] When solubilized hesperidin is added to an object containing a coloring agent or a vitamin, it absorbs ultraviolet rays such as sunlight or fluorescent light, and has an effect of preventing destruction of the coloring agent or the vitamin. Further, since the dissolved solubilized hesperidin is colorless and transparent, the color tone of the object does not change by adding it. Therefore, a large amount of solubilized hesperidin can be added to the object.

[0034]

[Brief description of the drawings]

FIG. 1 shows the absorption spectra of solubilized hesperidin and solubilized rutin.

FIG. 2: Comparison of HPLC patterns before and after β-amylase treatment of hesperidin glycoside Left diagram; right diagram before β-amylase treatment; after β-amylase treatment A; hesperidin triglyceride B; hesperidin diglyceride C; hesperidin monoglyceride D; hesperidin

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Yoshinobu Terada 1-30-4 Nori, Nishiyodogawa-ku, Osaka-shi, Osaka (56) References JP-A-3-7593 (JP, A) JP-A-4-13691 (JP) , A) JP-B-36-10693 (JP, B1) (58) Field surveyed (Int. Cl. ⁷ , DB name) A23L 1/27-1/305

Claims (6)

(57) [Claims] 1. The hesperidin at the 4-position of glucose
Compounds in which glucose is linked by α - 1,4 bonds
Glucose is further added to α-position at position 4
A method for stabilizing a food or the like containing a coloring agent and / or a vitamin, which comprises adding a solubilized hesperidin, which is a mixture of several bound ones . 2. The method for stabilizing a food or the like containing a coloring agent and / or a vitamin according to claim 1, wherein the solubilized hesperidin is α-glucopyranosyl hesperidin. 3. A strain A2-5a of which the solubilized hesperidin belongs to the genus Bacillus (Funded Deposit F of the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology).
CG collected from a culture of ERM P-13864)
The method for stabilizing a food or the like containing a colorant and / or a vitamin according to claim 1, wherein the method is produced using Tase. 4. In the position 4 of glucose of naringin
Compound in which glucose is bound by α-1,4 bond and its compound
Glucose at α- 1,4 bond at position 4 of glucose
Adding a solubilized naringin, which is a mixture of one to several dozen or so, and / or a coloring agent and / or
Or a method for stabilizing foods containing vitamins 5. The method for stabilizing a food or the like containing a coloring agent and / or a vitamin according to claim 4 , wherein the solubilized naringin is α-glucopyranosyl naringin. 6. A strain A wherein the solubilized naringin is Bacillus.
2-5a (Deposited FE of the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology)
CGT collected from cultures of RM P-13864)
A method for stabilizing a food or the like containing a colorant and / or a vitamin according to claim 4 , characterized in that the food is produced using ase.