JP3536138B2 - Prevention of crystal precipitation of hesperidin and prevention of white turbidity in liquid foods containing tangerines or tangerine juice - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preventing crystal precipitation of an aqueous solution containing hesperidin and a method for preventing clouding of syrup containing mandarin orange or mandarin orange juice.

[0002]

2. Description of the Related Art Hesperidin is a kind of flavonoid, and is abundantly contained in citrus fruits, especially Unshu mandarin oranges. Hesperidin has a solubility of 0.002% by weight in water, but dissolves in an alkaline aqueous solution at 1% by weight or more. Hesperidin is contained in the transparent citrus juice and canned mandarin orange in an amount of 0.03 to 0.07% by weight, respectively. Therefore, if the transparent juice of Satsuma mandarin or the can of Satsuma mandarin orange is left for a while, crystals of hesperidin will precipitate out, causing cloudiness, and the commercial value thereof will be reduced.

Conventionally, in order to prevent cloudiness of canned mandarin oranges, methyl cellulose has been added or hesperidin has been decomposed by hesperidinase. But,
Methylcellulose is required to be labeled as a synthetic paste, so
It tends to be disliked by both manufacturers and consumers, and cannot be used for export products. Hesperidinase has a complicated treatment procedure, and the chlorine gas used for sterilizing water reduces the enzymatic activity of hesperidinase. Also, from the past,
Hesperidin was removed by a synthetic adsorbent in order to prevent cloudiness of the orange juice. However, from the viewpoint of nutrition when drinking, since hesperidin can be expected to have an effect as vitamin P, it is desirable to remove as much as possible. In addition, since components other than hesperidin are also removed, there is a problem in flavor.

The present inventors have invented a technique for producing solubilized flavonoids by glycosylating poorly water-soluble flavonoids (Japanese Patent Application No. 5-256141). According to this invention, hesperidin can be glycosylated to produce solubilized hesperidin.

[0005]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned drawbacks of the prior art in preventing crystal precipitation of a solution containing hesperidin, preventing clouding of canned mandarin oranges and clouding of transparent orange juice drinks. Is an issue.

[0006]

The hesperidin to which the present invention is applied is rutinose (L-rhamnosyl- (α1) at the 7-position hydroxyl group of hesperitin (5,7,3'-trihydroxy-4'-methoxyflavanone). → 6) -Glucose) is β-bonded.

The solubilized hesperidin used in the present invention has glucose α- at the 4-position of glucose of hesperidin.
It is a compound bound by 1,4 bonds, each compound in which 1 to 10 or more glucose are sequentially bound by α-1,4 bonds to the 4-position of the glucose, or a mixture thereof.

Solubilized hesperidin can be produced by reacting glycosyltransferase with hesperidin. For example, C
GTase, namely 1,4-α-D-glucan;
4-α-D- (1,4-glucano) -trans
Ferase (EC 2.4.1.19) can be used for the production (Japanese Patent Application No. 5-256141).
issue). Any enzyme may be used for production as long as it is a glycosyltransferase. However, if an enzyme unstable in the alkaline region is used, the enzyme is easily deactivated and the yield of solubilized hesperidin is significantly reduced. Therefore, an alkali-resistant enzyme is preferably used.

A2 belonging to the genus Bacillus developed by the present inventors
-5a (Deposit of Bacteria, FERM P, Institute of Biotechnology, AIST)
-13864, hereinafter referred to as the present strain), novel CGTase collected from the culture retains high activity in the alkaline region and is therefore preferably used. The culture conditions of this strain and the method of collecting the enzyme are not special.

The conditions for the glycosyl transfer to hesperidin using this novel CGTase are the same as in the conventional method except that they are performed in the alkaline region, and are not special conditions. The pH is preferably 8 to 11, the temperature is 20 to 75 ° C., the donor concentration is 0.1 to 30%, preferably 1 to 20%, and the hesperidin concentration is 0.1 to 5%, preferably 0.5 to 2
%, The enzyme is 0.1 to 100 units / ml to start the reaction. The longer the reaction time, the better as long as the enzyme activity is sustained. The reaction is stopped by heating at 100 ° C. for 5 minutes.

This reaction solution was added to Amberlite XAD-16.
After passing through a resin and washing with water, the fraction eluted with 50% by weight of ethanol is concentrated to obtain a solubilized hesperidin in a liquid form or in a powder form by drying. The solubilized hesperidin obtained here is a mixture of each compound in which 1 to 10 or more glucoses are sequentially bound to the 4-position of glucose of hesperidin by α-1,4 bonds. The solubilized hesperidin thus obtained was further subjected to gel filtration (Sephadex LH
As a result of the separation according to 20), glucose is attached to the 4-position of glucose of hesperidin with an α-1,4 bond at 1 position.
One, two, and three are combined.

As the donor, cyclodextrin, various oligosaccharides including maltooligosaccharide, and α-1,4 glucan having α-1,4 bond such as starch are used.

The mandarin oranges or syrup containing mandarin orange juice used in the present invention include canned mandarin oranges and mandarin orange juice.
Canned mandarin oranges are those specified by the Japanese Agricultural Standards. That is, the outer skin and seeds of mandarin orange fruits are removed, peeled by an acid or alkali method, placed in a metal can for food canning, canned syrup is added thereto, the cans are closed, and then sterilized. Wenzhou mandarin orange is particularly preferable as the mandarin orange fruit, but any fruit containing hesperidin such as summer mandarin orange, Iyo mandarin orange, and navel orange may be used. Syrup in canned mandarin orange is an aqueous solution containing sugar as a main component. In addition, glucose, sodium cyclamate, artificial sweeteners such as saccharin, and acidulants such as citric acid may be included.

The mandarin orange transparent fruit juice used in the present invention is a natural fruit juice obtained by converting the natural fruit juice into a transparent solution by a method such as Keisei algal filtration or ultrafiltration. Also included are fruit juice drinks, soft drinks containing fruit juice, and fruit drinks containing fruit granules in which cloudiness may be a problem. The natural fruit juice and fruit juice drink, soft drink containing fruit juice, and fruit drink containing fruit granules as referred to herein are as defined in the Japanese Agricultural Standards. In other words, natural juice is 100% by weight, and fruit juice is 50% by weight.
The above content of less than 100% by weight, the soft drink containing fruit juice contains 10% by weight or more and less than 50% by weight of fruit juice,
The fruit drink containing fruit granules refers to one having a fruit juice content of 10% by weight or more and less than 100% by weight and a fruit particle content of 5% by weight or more and less than 30% by weight. The raw material of the fruit juice is mainly Wenzhou mandarin orange, but any fruit containing hesperidin such as summer mandarin orange, Iyo mandarin orange, and navel orange may be used. In addition to the pulp of the fruit juice raw material, the fruit grains containing the fruit juice may be other than the juice raw material such as peach pulp and pineapple pulp.

In the present invention, "to add" means to prepare solubilized hesperidin as a powder or to prepare and add a solution having a concentration of 100 to 10000 times the use concentration. The objects to be added are an aqueous solution containing hesperidin, which causes problems of crystal precipitation and cloudiness due to hesperidin, syrup in canned mandarin oranges, and clear orange juice. The addition amount is 0.001% by weight to 10% by weight in an aqueous solution containing hesperidin.
For canned mandarin orange syrup, 0.001% by weight to 1
%, Preferably 0.003 to 0.1% by weight. In the case of mandarin orange juice, 0.01% by weight to 10%
%, Preferably 0.05 to 0.1% by weight.

[0016]

Function Hesperidin is soluble in alkaline aqueous solution, but has low solubility in neutral to acidic aqueous solution. Therefore, when the pH of the aqueous solution in which hesperidin is dissolved becomes acidic, hesperidin precipitates as crystals and the aqueous solution becomes cloudy. The cloudiness of the solution due to the crystallization of hesperidin is influenced by the pulp quality, protein, etc. in the solution containing hesperidin, and in the presence of these, cloudiness due to the crystallization of hesperidin is promoted. Solubilized hesperidin inhibits hesperidin crystallization in proportion to its concentration. for that reason,
The amount of solubilized hesperidin added is appropriately determined depending on the degree of formation of hesperidin crystals in an aqueous solution containing hesperidin. That is, as described above, the addition amount is increased in the presence of pectic substances and proteins. Also, solubilized hesperidin is tasteless and the color tone is colorless to pale yellow, but when added to a high concentration, this slight color tone cannot be ignored.
Also, a slight change in flavor can be felt. Therefore, the maximum addition amount is determined by the range in which the changes are allowed depending on the addition target.

[0017]

【Example】

(Example 1) 0.001% by weight of solubilized hesperidin was added to an aqueous solution containing 0.12% by weight of hesperidin.
%, 0.05% by weight was added to the test sections 1, 2,
It was set to 3. A group containing no solubilized hesperidin was used as a control group. For hesperidin, a high-concentration solution (2% by weight of 0.1N sodium hydroxide aqueous solution) was prepared, added to have the above concentration, and adjusted to pH 3.0 with 1N hydrochloric acid. As the solubilized hesperidin, one in which one glucose was bound with α-1,4 bond at the 4-position of glucose of hesperidin (hereinafter referred to as hesperidin monoglucoside) was used to prepare a 10% by weight aqueous solution, and each test described above was performed. It was added to make the concentration. Each test solution was allowed to stand at 4 ° C, a part of the test solution was taken every day to remove the insoluble fraction by centrifugation, and the hesperidin (hereinafter referred to as soluble hesperidin) dissolved in water in the supernatant was Quantitative analysis was carried out by HPLC shown in. HPLC analysis conditions Column: ODS Solvent: Acetonitrile / water = 20/80 Flow rate: 0.5 ml / min Column temperature: 40 ° C. Detection: 280 nm

The results of measurement of the amount of soluble hesperidin in the aqueous solution of hesperidin are shown in FIG. Test start 1 in control section
The soluble hesperidin amount decreased to 0.02% by weight after one day, while in test area 1, it was 0.05 after one day from the start of the theory.
%, After 3 days was 0.03% by weight, and in test section 2, it was 0.07% by weight after 1 day and 0.04% by weight after 3 days.
Further, in the test area 3, even after 3 days from the start of the test, 0.
1% by weight of soluble hesperidin remained. There is a relationship shown in Formula 1 between the amount of hesperidin added to the aqueous solution at the start of the test (total hesperidin amount), the hesperidin crystal amount, and the soluble hesperidin amount in the aqueous solution. [Formula 1] (Total hesperidin amount) = (Hesperidin crystal amount) + (Soluble hesperidin amount) Therefore, the decrease amount of the soluble hesperidin in the aqueous solution corresponds to the production amount of hesperidin crystals. Therefore, the above results revealed that soluble hesperidin has an effect of preventing crystal formation of hesperidin in an aqueous solution.

Example 2 As a canned tangerine model syrup, solubilized hesperidin was added to a solution containing 16.7% by weight of sucrose, 3% by weight of glucose, 0.63% by weight of citric acid and 0.03% by weight of hesperidin. 001% by weight, 0.0
05% by weight and 0.01% by weight were added to make test groups 1, 2, and 3. Here, the hesperidin concentration was determined from the average concentration in canned citrus syrup. The solubilized hesperidin has α at the 4-position of glucose of hesperidin.
A 10% by weight aqueous solution was prepared by using one having a 1,4 bond and one glucose bond with the α-1,4 bond at the 4-position of glucose (hereinafter referred to as hesperidin diglucoside). It was added so that it might become a test concentration. These were allowed to stand at room temperature and the cloudiness of the solution was observed after 3 days and 30 days, and at the same time, the amount of hesperidin in the solution was quantified by the above-mentioned HPLC (ODS).

[0020]

[Table 1]

Table 1 shows the observation results of white turbidity of canned orange syrup. In this test, a group containing no solubilized hesperidin was used as a target group. Three days after the start of the test, white turbidity was observed in all the five repeated tests in the target group, but no white turbidity was observed in the test groups 1, 2, and 3 containing solubilized hesperidin. The same result was obtained after 30 days.

FIG. 2 shows the results of measuring the concentration of hesperidin in canned syrup of mandarin orange by HPLC (ODS). Three
After a day, the hesperidin concentration was decreased in the control group, but in the test groups 1, 2, and 3, the hesperidin concentration at the start of the test was almost maintained. From this, it was shown that solubilized hesperidin inhibited crystallization of hesperidin in syrup, and as a result, cloudiness did not occur. Moreover, the same result was obtained after 30 days, and it was confirmed that the effect of solubilized hesperidin was maintained.

(Comparative Example 1) β-cyclodextrin (β-), which is known to have an effect of preventing clouding of canned syrup of mandarin oranges
CD) was compared. A test section in which 0.1% by weight of β-CD (Note 1) was added to the canned orange syrup,
The effect of preventing white turbidity was compared between the test group to which 0.1% by weight of solubilized hesperidin was added. Hesperidin monoglucoside was used as the solubilized hesperidin, and each powder of the solubilized hesperidin and β-CD was added so as to have the above-mentioned test concentration. After preparing each mandarin orange canned syrup, it was left at room temperature, and the cloudiness of the syrup was observed after 3 days and 30 days, and at the same time, the amount of hesperidin in the mandarin orange canned syrup was measured as described above.
It was measured by PLC (ODS). At this time, a mandarin orange canned syrup containing no β-CD and solubilized hesperidin was used as a control. (Note 1) Up to about 0.04% by weight of β-CD, it is known that the solubilization of hesperidin is promoted in proportion to its concentration. However, solubilization of hesperidin is hardly changed at higher concentrations. The concentration of β-CD in this test is an amount necessary and sufficient for observing the hesperidin solubilizing effect, that is, the cloudiness preventing effect.

[0024]

[Table 2]

Table 2 shows the results of observation of white turbidity of syrup. In the control group, white turbidity was observed in all 5 treatments after 3 days, and in the β-CD addition group, 1 treatment out of 5 treatments after 3 days,
After 30 days, white turbidity was observed in 3 out of 5 treatments. In contrast, in the solubilized hesperidin-added group, no cloudiness was observed even after 30 days.

Further, the concentration of hesperidin in canned syrup of mandarin orange was measured (FIG. 3).
After the day and 30 days after the addition of β-CD, 0.
Although it was reduced from 035% by weight to 0.01% by weight, the concentration of hesperidin was not reduced in the solubilized hesperidin addition group.

Example 3 As an orange juice model solution, 1% by weight of citric acid, 0.67% by weight of tripotassium citrate, 0.1% by weight of benzoic acid, 0.2% by weight of bectin,
Solubilized hesperidin in a solution containing 0.15% by weight of hesperidin 0.01% by weight, 0.05% by weight, 0.1% by weight
In addition, test sections 1, 2, and 3 were prepared. Let these stand at room temperature,
The cloudiness of the solution was measured by the turbidity at a wavelength of 660 nm. As solubilized hesperidin, hesperidin diglucoside was used.
A 0% by weight aqueous solution was prepared and added to each of the above test concentrations.

The results of measuring the cloudiness of the solution over time are shown in FIG.
It was shown to. In this test, a group containing no solubilized hesperidin in the solution was used as a control group. Start of test 1 in control section
The turbidity of the solution increased after 2 hours and reached the maximum after 2 hours, but the turbidity reached maximum after 3 hours and 24 hours in test sections 1 and 2, respectively, and even after 24 hours in test section 3. No cloudiness was observed.

When the concentration of hesperidin in the orange juice solution was measured by HPLC (ODS) in the same manner as described above (FIG. 5), the control group showed cloudiness of the solution and a decrease in the concentration of hesperidin. In 2 and 3, the decreasing rate of hesperidin concentration was slower than that in the control group.
From this, it was shown that the solubilized hesperidin was one that prevented or delayed the occurrence of white turbidity as a result of inhibiting or delaying the crystallization of hesperidin in the solution.

(Example 4) Canned mandarin orange was used as No. 4 can, and 290 g of Satsuma mandarin pulp and solubilized hesperidin were 0.02% by weight, 0.05% by weight and 0.10% by weight in one can.
Injected 160 g of the added canned syrup (sugar content 14 to 15, pH 3.7 to 3.8) was tested in Test Groups 1, 2, and 3.
And These were wound in the usual manner, sterilized and cooled, and then appropriately shaken and stored at room temperature. The solubilized hesperidin is obtained by adding one or two glucose bonds at the 4-position of glucose of hesperidin with α-1,4 bond to 1:
What was contained in 1 (hereinafter referred to as mixed solubilized hess 0 lysine) was added as a powder to each of the above test concentrations.
In addition, groups without solubilized hesperidin and with 0.05 wt% of hesperidinase were designated as control groups 1 and 2, respectively. The amount of hesperidin in canned syrup is
It was 0.03% when measured by LC. These canned mandarin oranges were opened 3 months after production, and the transparency of the syrup was examined by the number of readings of a cylinder type turbidimeter officially used by the Canning Inspection Association.

The measurement results of canned syrup turbidity are shown below. The smaller the number, the more cloudy the measured value. -Control section 1 --- 74mm-Control section 2 --- 200mm-Test section 1--200mm or more-Test section 2 --- 200mm or more-Test section 3 --- 200mm or more

In the control group 1 containing no solubilized hesperidin, white turbidity was observed after 3 months, whereas in the control group 2 containing hesperidinase and the test groups 1, 2 and 3 containing solubilized hesperidin, almost clouding occurred. I couldn't see it. Further, in comparison between the control section 2 and the test sections 1, 2, and 3, the test sections 1, 2, and 3 to which the solubilized hesperidin was added had higher transparency. From the above results, it was found that solubilized hesperidin has an effect of preventing clouding of canned syrup of mandarin orange, and that effect is superior to that of hesperidinase.

(Example 5) Natural fruit juice of 100% Satsuma mandarin orange was centrifuged to remove the precipitate, and the supernatant was treated by ultrafiltration (MW 30,000) to obtain a high concentration solution of hesperidin. The hesperidin concentration was adjusted to 0.03% by weight with (1% by weight 0.1N sodium hydroxide aqueous solution) to obtain transparent orange juice. Solubilized hesperidin was added to the transparent orange juice in an amount of 0.01% by weight and 0.1% by weight to prepare test sections 1 and 2. These were allowed to stand at 4 ° C., and the cloudiness of the solution was observed 5 and 30 days after the start of the test. In addition, a group containing no solubilized hesperidin was used as a control group, and three repeated tests were conducted. As the solubilized hesperidin, a mixed solubilized hesperidin was used to prepare a 10% by weight aqueous solution, and the solutions were added to each of the above-mentioned test concentrations.

The result of observing the cloudiness of transparent orange juice is shown in FIG. Turbidity began to rise in the control plots 5 days after the start of the test, and after 30 days the turbidity was 10 times that at the start of the test, but in test plots 1 and 2 the turbidity increased 5 days after the start of the test. However, even after 30 days, the increase in the turbidity was 5 times higher than that at the start of the test in test section 1 and about 3 times that at the start of the test in test section 2.

[0035]

By adding solubilized hesperidin to an aqueous solution containing hesperidin, crystallization of hesperidin and accompanying clouding of the aqueous solution can be prevented.

Canned mandarin oranges can easily and more effectively prevent clouding without requiring a complicated operation such as enzyme treatment. Further, the solubility is higher than that of methyl cellulose, and the viscosity of the solution is not increased.

In the transparent orange juice, turbidity can be prevented by a simple method of adding solubilized hesperidin to the juice without requiring a complicated operation such as a synthetic adsorbent.
In addition, since it is not necessary to remove hesperidin, it is possible to produce transparent mandarin orange juice which is nutritionally superior to the conventional method. Also, there is no problem in flavor.

[0038]

[Brief description of drawings]

[Figure 1] Quantification of hesperidin amount in hesperidin aqueous solution ▲ Black square ▼: Solubilized hesperidin is not added (control group) ◇: Solubilized hesperidin 0.001% is added (test group 1) □: Solubilized hesperidin 0.01% Addition (test section 2) ○: Solubilized hesperidin 0.05% addition (test section 3)

[Fig. 2] Determination of hesperidin in canned orange syrup model syrup Control group: solubilized hesperidin-free test group 1: solubilized hesperidin 0.001% added test group 2: solubilized hesperidin 0.005% added test group 3: solubilized hesperidin 0.01% added

[Fig. 3] Quantitative solubilization of hesperidin in canned tangerine model syrup Comparison between hesperidin and β-CD Control group: Solubilized hesperidin-free test group 1: 0.1% Solubilized hesperidin-added test group 2: 0.1% β-CD added

Fig. 4 Turbidity of orange juice model solution at a wavelength of 660 nm ▲: Black square ▼: Solubilized hesperidin is not added (control group) ◇: Solubilized hesperidin 0.01% is added (test group 1) □: Solubilized hesperidin 0. 05% addition (test section 2) ○: Solubilized hesperidin 0.1% addition (test section 3)

[Fig. 5] Quantification of hesperidin in mandarin juice model solution ▲ Black square ▼: Solubilized hesperidin is not added (control group) ◇: Solubilized hesperidin 0.01% is added (test group 1) □: Solubilized hesperidin 0.05 % Addition (test section 2) ○: Solubilized hesperidin 0.1% addition (test section 3)

[FIG. 6] Turbidity of transparent orange juice at wavelength of 660 nm ▲ Black square ▼: Solubilized hesperidin is not added (control group) ◇: Solubilized hesperidin 0.01% is added (test group 1) ○: Solubilized hesperidin 0.1 % Addition (test area 2)

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroshi Takai 1-30-4 Nori, Nishiyodogawa-ku, Osaka-shi, Osaka (72) Inventor Yoshinobu Terada 1-30-4 Nori, Nishiyodogawa-ku, Osaka-shi, Osaka (56) References JP-A-2-86757 (JP, A) JP-A-49-485 (JP, A) JP-A-49-11900 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) A23L 2/70

Claims (9)

(57) [Claims] 1. A method for preventing the precipitation of crystals of hesperidin, which comprises adding solubilized hesperidin to an aqueous solution containing hesperidin. 2. A solubilized hesperidin is a compound in which glucose is bound to the 4-position of glucose of hesperidin with an α-1,4 bond, and one glucose is further sequentially bound to the 4-position of glucose with an α-1,4 bond. 10. The method for preventing crystal precipitation of hesperidin according to claim 1, wherein each compound is composed of 10 to 10 bound compounds, or a mixture thereof. 3. Solubilized hesperidin is a compound in which glucose is bound to the 4-position of glucose of hesperidin with an α-1,4 bond, and one glucose is further sequentially bound to the 4-position of glucose with an α-1,4 bond. The compound for preventing hesperidin crystal precipitation according to claim 1, wherein each compound is a compound in which two are bonded, or a mixture thereof. 4. The amount of solubilized hesperidin added is 0.001.
The method for preventing crystal precipitation of hesperidin according to any one of claims 1 to 3, which is from 10% by weight to 10% by weight. 5. A method for preventing clouding of mandarin orange or syrup containing mandarin orange juice, which comprises adding solubilized hesperidin. 6. A solubilized hesperidin is a compound in which glucose is bound to the 4-position of glucose of hesperidin with an α-1,4 bond, and one glucose is further sequentially bound to the 4-position of glucose with an α-1,4 bond. 10. The method for preventing clouding of syrup according to claim 5, wherein each compound is composed of 10 to 10 bound compounds, or a mixture thereof. 7. Solubilized hesperidin is a compound in which glucose is bound to the 4-position of glucose of hesperidin with an α-1,4 bond, and one glucose is further sequentially bound to the 4-position of glucose with an α-1,4 bond. The compound for preventing turbidity of syrup according to claim 5, wherein each compound is a compound in which two are bonded, or a mixture thereof. 8. The method for preventing white turbidity of syrup according to claim 5, wherein the syrup is a syrup containing mandarin orange and the amount of solubilized hesperidin added is 0.001% by weight to 1% by weight. 9. The syrup is a syrup containing mandarin orange juice, and the added amount of solubilized hesperidin is 0.01% by weight.
To 10% by weight, the method for preventing white turbidity of syrup according to claim 5.