JP5985229B2 - Process for producing sugar adducts of poorly water-soluble polyphenols - Google Patents

Description

  The present invention relates to a method for producing a sugar adduct of poorly water-soluble polyphenols.

Recently, various materials having physiological functions have been proposed, and many health foods containing these have been put on the market. Among them, polyphenols are known to have an antioxidant power and are expected to have effects such as anti-arteriosclerosis, anti-allergy and blood flow enhancement, and thus are recognized as important ingredients in health foods.
However, many polyphenols have poor water solubility, and it is difficult to use them for aqueous foods such as soft drinks.

  Therefore, a technique for solubilizing poorly water-soluble polyphenols in water has been studied, and sugar adducts obtained by adding sugars such as glucose to polyphenols have been proposed. For example, the solubility of hesperidin in water at 25 ° C. is only 0.02 mg / g, but α-glucosyl hesperidin in which glucose is bound to hesperidin has a high solubility in water at 25 ° C. of 200 mg / g or more. Become. Furthermore, the sugar addition product of polyphenol has advantages such as exerting physiological functions equivalent to those of polyphenol.

  As a method for producing a sugar addition product of polyphenol, for example, sugar transfer to a solution containing rutin or hesperidin in a suspended state or dissolved at an alkaline pH exceeding 7.0, and a partial hydrolyzate of starch, etc. A method of allowing an enzyme to act (Patent Documents 1 and 2), a flavonoid glycosyl transfer method (Patent Document 3) in which a flavonoid is dissolved in a thickened polysaccharide solution adjusted to pH 8 to 10 and a cyclodextrin synthase is allowed to act; There has been reported a flavonoid glycoside production method (Patent Document 4) in which the solubilization is performed in an alkaline region of pH 8 or higher and / or cyclodextrin is added, and a cyclodextrin synthase is allowed to act in the alkaline region.

JP-A-3-27293 JP-A-3-7593 JP-A-10-101705 JP-A-7-107972

However, as disclosed in Patent Documents 1 to 4, the method of reacting with a glycosyltransferase after dissolving polyphenol in an alkaline aqueous solution is low in stability of the polyphenol in the alkaline region and easily decomposed, and also neutralizes the alkali. And a desalting step is necessary, and there is a concern that the manufacturing process may become complicated. In addition, as in Patent Documents 1 and 2, the method of allowing glycosyltransferase to act on a solution in which a poorly water-soluble polyphenol is suspended has a sufficient conversion rate because the amount of polyphenol dissolved in the transglycosylation reaction solution is small. It may not be possible.
Accordingly, an object of the present invention is to provide a method for efficiently producing a sugar adduct from poorly water-soluble polyphenols.

  The present inventors have intensively studied in view of the above problems, and after heat-treating a poorly water-soluble polyphenol having a rhamnoside structure in a specific temperature range, the enzyme having a rhamnosidase activity is allowed to act within a specific time. When rhamnose of polyphenols is eliminated and then glycosyltransferase is allowed to act on the reaction solution, the dissolution concentration of poorly water-soluble polyphenols having a rhamnoside structure is greatly increased and the rhamnose elimination reaction is maintained while maintaining a high concentration. It was found that a polyphenol sugar adduct having excellent water solubility can be obtained in a high yield because the sugar transfer reaction proceeds at a high concentration.

That is, the present invention includes the following steps (1), (2) and (3):
(1) A step of heat-treating the poorly water-soluble polyphenols (A) having a rhamnoside structure at 100 to 180 ° C. in the presence of an aqueous medium to obtain a heat treatment liquid,
(2) A step of starting a rhamnose elimination reaction within 300 minutes after the heat treatment to obtain a reaction solution containing the poorly water-soluble polyphenols (A ′) from which rhamnose has been removed, and (3) A step of adding a sugar donor (B) and a glycosyltransferase to the obtained reaction solution to obtain a sugar adduct of a poorly water-soluble polyphenol from which rhamnose has been removed by a sugar transfer reaction. A method for producing a sugar adduct of polyphenols is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the conversion rate of a rhamnose elimination reaction and the yield of a polyphenol sugar adduct can be improved, and the sugar adduct of polyphenols excellent in solubility can be manufactured with a high yield.

  Step (1) in the method of the present invention is a step of obtaining a heat-treated liquid by heat-treating the poorly water-soluble polyphenols (A) having a rhamnoside structure at 100 to 180 ° C. in the presence of an aqueous medium.

  In the present specification, “poorly water-soluble polyphenols” refers to polyphenols having a log P value of −1.0 to 4.0. The slightly water-soluble polyphenols preferably have a log P value of -0.5 to 3.5. The log P value is a value obtained by taking the common logarithm of the distribution coefficient between 1-octanol / water and is an index indicating the hydrophobicity of an organic compound. The larger the value, the higher the hydrophobicity. The log P value of polyphenol can be measured by a flask shaking method described in Japanese Industrial Standard Z7260-107. Details are described in the examples. Further, it is preferably applied to those having a solubility in water at 25 ° C. of 5 g / L or less, more preferably 2 g / L or less, further 1 g / L or less, further 0.5 g / L or less, and further 0.1 g. / L or less is preferable.

The poorly water-soluble polyphenols (A) having a rhamnoside structure used in the present invention are polyphenols having a rhamnoside structure having a log P value of -1.0 to 4.0. As the raw material polyphenols (A) having a rhamnoside structure, phenolic substances having one or more, more preferably two or more hydroxy groups bonded to the benzene ring and having a rhamnoside structure can be preferably applied. For example, plant-derived flavonoids, tannins, phenolic acids and the like can be mentioned. Examples of the poorly water-soluble polyphenols having a rhamnoside structure that can be more preferably applied include those selected from flavonols, flavanones, flavones, isoflavones, and phenolcarboxylic acids having a rhamnoside structure.
Specific examples include rutin, quercitrin, myricitrin, hesperidin, neohesperidin, naringin, or derivatives thereof. Examples of the derivatives include acetylated products, malonylated products, and methylated products. Among these, flavonols and flavanones having a rhamnoside structure are preferably applicable because of low solubility of the raw materials, flavanones having a rhamnoside structure are more preferable, and rutin and hesperidin are more preferable. The poorly water-soluble polyphenols having the rhamnoside structure may be one kind or a mixture of two or more kinds.

  In the present specification, the aqueous medium refers to water and an aqueous solution of an organic solvent. Examples of water include tap water, distilled water, ion exchange water, and purified water. The organic solvent is not particularly limited as long as it is uniformly mixed with water. As the organic solvent, alcohol having 4 or less carbon atoms is preferable, methanol and ethanol are more preferable, and ethanol is more preferable from the viewpoint of being applicable to foods. When an aqueous solution of an organic solvent is used as the aqueous medium, the concentration of the organic solvent in the aqueous solution is preferably 0.1 to 80% by mass, more preferably 1 to 70% by mass, and still more preferably 5 to 60% by mass.

  The pH of the aqueous medium is preferably 3 or more and less than 7, more preferably 3.5 to 6.9, and more preferably 4 to 6.8 from the viewpoint of the stability of the poorly water-soluble polyphenols (A) having a rhamnoside structure as a raw material. Is more preferable.

  Since the poorly water-soluble polyphenols (A) having a rhamnoside structure as a raw material have low solubility in water, it is preferable that they be dispersed in an aqueous medium and exist in a slurry state. The content of the poorly water-soluble polyphenols (A) having a rhamnoside structure as a raw material in the aqueous medium varies depending on the kind of the poorly water-soluble polyphenols, but is 0.1 to 100 g / L from the viewpoint of the fluidity of the slurry. Is preferable, 0.5-50 g / L is more preferable, 0.7-20 g / L is still more preferable, and 0.7-10 g / L is still more preferable.

The method for heat-treating the poorly water-soluble polyphenols (A) having a rhamnoside structure as a raw material in the presence of an aqueous medium is not particularly limited, and known methods can be applied.
Although the temperature of heat processing is 100-180 degreeC, 110-170 degreeC is more preferable, 120-160 degreeC is still more preferable, 120-150 degreeC is still more preferable. A significant improvement in solubility is achieved at 100 ° C. or higher, and the stability of the poorly water-soluble polyphenols (A) having a raw rhamnoside structure at 180 ° C. or lower is ensured. Examples of the heating means include water vapor and electricity.

  The pressure during the heat treatment is preferably 0 to 10 MPa, more preferably 0.1 to 8 MPa, further preferably 0.1 to 6 MPa, further preferably 0.2 to 6 MPa, and further preferably 0.2 to 4 MPa in terms of gauge pressure. Preferably, 0.25 to 2 MPa is more preferable, 0.3 to 1.5 MPa is further preferable, and 0.3 to 0.6 MPa is further preferable. Moreover, it is preferable to set it more than the saturated vapor pressure of water. Pressurization higher than the saturated vapor pressure may be adjusted by a back pressure valve, or gas may be used. As the gas used, for example, an inert gas is preferable, and nitrogen gas and helium gas are more preferable.

  The heat treatment can be performed by any method such as a batch method, a semi-batch method, and a flow-type treatment method. Among these, the flow-type processing method is preferable in that the processing time can be easily controlled.

The heat treatment time is from 0.1 to 30 minutes in the temperature range of the heat treatment from the viewpoint of improving the solubility and heat stability of the poorly water-soluble polyphenols (A) having a rhamnoside structure as a raw material. Preferably, it is further 0.2 to 15 minutes, and further preferably 0.5 to 8 minutes.
In the case of using the flow treatment method, the heat treatment time is an average residence time calculated by dividing the volume of the high-temperature high-pressure part of the processor by the supply rate of the aqueous medium.

  After the heat treatment, it is preferable from the viewpoint of preventing thermal degradation of the polyphenol that the obtained heat treatment liquid is cooled to 90 ° C. or lower, preferably 50 ° C. or lower, more preferably 30 ° C. or lower. When cooling, the heat treatment liquid may be mixed and stirred.

  The cooling rate of the heat treatment liquid calculated from the time required to decrease from the heat treatment temperature to 90 ° C. is 0.2 ° C./s or more, further 0.5 ° C./s or more, 1 ° C./s or more, and further 3 It is preferably at least 5 ° C / s, more preferably at least 5 ° C / s. As the cooling rate increases, the amount of the poorly water-soluble polyphenols (A) having a rhamnoside structure as a raw material can be improved. For this reason, the upper limit of the cooling rate is not particularly defined, but is preferably 100 ° C./s or less, and more preferably 50 ° C./s or less.

  Furthermore, it is preferable to carry out the step of removing the solid that remains without being dissolved from the heat treatment liquid, from the viewpoint of increasing the solubility of the resulting poorly water-soluble polyphenols sugar adduct. The method for removing the solid is not particularly limited, and can be performed by, for example, centrifugation, decantation, or filtration. The removal of the solid is preferably performed before the following step (2).

In the method of the present invention, the heat treatment may be performed in the presence of the solubilizer (C). In the heat treatment, the mass ratio of the solubilizer (C) to the total of the poorly water-soluble polyphenols (A) having the rhamnoside structure and the solubilizer (C) [(C) / ((A) + (C) )] Is less than 0.1 to improve the conversion rate of the raw water-insoluble polyphenols (A) having a rhamnoside structure to the water-insoluble polyphenols (A ′) from which rhamnose has been eliminated. In view of reducing the purification load of the reaction product.
That is, the relationship between the poorly water-soluble polyphenols (A) having a rhamnoside structure as a raw material and the solubilizer (C) is
0 ≦ (C) / ((A) + (C)) <0.1
It can be expressed as.
From the viewpoint of reducing the purification load of the reaction product, the mass ratio is preferably 0.09 or less, more preferably 0.07 or less, further preferably 0.05 or less, further preferably 0.04 or less, 0 That is, a condition in which the solubilizer (C) is not present is particularly preferable.

  As the solubilizer (C), those having higher water solubility than the poorly water-soluble polyphenols (A) having a rhamnoside structure as a raw material are preferable, and a sugar adduct of the poorly water-soluble polyphenols (A) having a rhamnoside structure is preferable. Preferably, hesperetin sugar adducts such as glucosyl hesperidin and glucosyl hesperetin are more preferably used.

  In the step (2) of the present invention, the rhamnose elimination reaction is started within 300 minutes after the heat treatment for the heat treatment liquid obtained in the step (1), and the poorly water-soluble polyphenols (A This is a step of obtaining a reaction solution containing ').

The rhamnose elimination reaction is a reaction in which rhamnose is eliminated from the poorly water-soluble polyphenols (A) having a rhamnoside structure to obtain poorly water-soluble polyphenols (A ′) from which rhamnose is eliminated.
The rhamnose elimination reaction is started by adding an enzyme having rhamnosidase activity to the heat treatment solution and setting the reaction temperature to express the rhamnosidase activity.

In this specification, “within 300 minutes” after the heat treatment refers to the time from the time when the heat treatment is completed, that is, the time when the heat treatment liquid is lowered to less than 100 ° C. to the start of the rhamnose elimination reaction.
The time from the heat treatment to the start of the rhamnose elimination reaction is preferably 0.1 to 300 minutes, more preferably 0.1 to 180 minutes, and further preferably 1 to 120 minutes, from the viewpoint of improving the rhamnose elimination reaction rate. Preferably, 1 to 60 minutes is preferable.

The enzyme having the rhamnosidase activity used in the step (2) may be any enzyme having an activity to desorb rhamnose from the rhamnoside structure. For example, a naringinase preparation which is an enzyme desorbing rhamnose from naringin, rhamnose from hesperidin Examples include hesperidinase preparations and cellulase preparations that are enzymes to be eliminated.
The origin of the enzyme having rhamnosidase activity is not limited, and those derived from animals, plants, microorganisms, etc. can be used. Further, it may be an artificial enzyme by gene recombination technique, partial hydrolysis or the like.
The form of the enzyme is not particularly limited, and a dried product of enzyme protein, particles containing enzyme protein, liquid containing enzyme protein, and the like can be used.

  The amount of the enzyme having rhamnosidase activity varies depending on the type of enzyme used, the reaction conditions, the kind of poorly water-soluble polyphenols (A) having a raw material rhamnoside structure, and in the case of hesperidinase, for example, the poorly water-soluble material having a raw material rhamnoside structure 0.01-10 U is preferable with respect to 1 g of water-soluble polyphenols (A). Here, the amount of enzyme that generates 1 μmol of sugar per minute at 40 ° C. and pH 3.8 from hesperidin is defined as 1 unit (1 U).

  The conditions for the rhamnose elimination reaction in step (2) can be selected according to the characteristics of the enzyme used, the reaction temperature and the pH of the reaction solution. For example, when hesperidinase or naringinase is used, the pH is 3-7. It is preferable to adjust the pH to 4 to 6.5.

  The reaction temperature is preferably 10 to 80 ° C., more preferably 20 to 60 ° C., and more preferably 35 to 55 ° C. from the viewpoint of the stability of the enzyme having rhamnosidase activity and the improvement of the reaction rate of the rhamnose elimination reaction. .

The reaction is completed by, for example, a method of inactivating the enzyme by heating the reaction solution or a method of separating and removing the enzyme from the reaction solution.
Although the reaction time varies depending on the type of enzyme having rhamnosidase activity, etc., the point of improving the rhamnose elimination rate of the poorly water-soluble polyphenols (A) having a rhamnoside structure, the poorly water-soluble polyphenols from which rhamnose is eliminated (described later) From the viewpoint of improving the productivity of the sugar adduct of A ′), for example, 0.5 to 120 hours are preferable, 1 to 100 hours, and further 2 to 20 hours are preferable.

  After completion of the step (2), the liberated rhamnose may be removed from the reaction solution by a resin adsorption method or an alcohol precipitation method. As the resin, synthetic adsorption resin Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) or the like is used. In the treatment method, 10 to 1000 ml of resin is added to 1 g of the raw material poorly water-soluble polyphenol before reaction to the reaction solution, and after passing through the column at 10 to 55 ° C., the resin is washed with water to obtain 30 to 80% ethanol water. To elute.

  According to the step (2), rhamnose was eliminated from the poorly water-soluble polyphenols (A) having a rhamnoside structure at a rhamnose elimination reaction rate of 50 to 100%, further 60 to 95%, and further 70 to 90%. Slightly water-soluble polyphenols (A ′) can be produced.

Step (3) in the method of the present invention is a slightly water-soluble polyphenol obtained by adding a sugar donor (B) and a glycosyltransferase to the reaction solution obtained in step (2) and releasing rhamnose by a sugar transfer reaction. This is a step of obtaining the sugar adduct.
In this sugar addition reaction, since the water-solubility of the poorly water-soluble polyphenols (A ′) from which rhamnose is eliminated is increased, there is no limitation on the reaction start time as in step (2).

The sugar donor (B) used in step (3) of the present invention is not particularly limited as long as it can donate the sugar described below to the poorly water-soluble polyphenols (A ′) from which rhamnose has been eliminated.
Specific examples of the sugar donor (B) include starch, dextrin, cyclodextrin, malto-oligosaccharide and other starch partial hydrolysates, xylooligosaccharide, fructan, arabinogalactan, pullulan, raffinose, and contents thereof. It is done.

  The amount of the sugar donor (B) used is a slightly water-soluble polyphenol from which rhamnose has been eliminated at the start of the transglycosylation reaction in order to improve the conversion rate of the poorly water-soluble polyphenols (A ′) from which rhamnose has been eliminated. The mass ratio (B) / (A ′) to the class (A ′) is preferably 1 to 30, more preferably 1.2 to 15, and further preferably 2 to 12.

The glycosyltransferase used in the step (3) of the present invention is not particularly limited as long as it is an enzyme having a sugar transfer activity with respect to the poorly water-soluble polyphenols (A ′) from which rhamnose has been eliminated. It can select suitably according to the kind of sugar donor (B).
Specific examples of glycosyltransferases include cyclomaltodextrin glucanotransferase, dextrin dextranase, amylosucrase, dextransucrase, α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, α-amylase, xylanase, pullulanase And arabinofuranosidase.

The origin of the glycosyltransferase is not limited, and those derived from all origins such as those derived from animals, plants, and microorganisms can be used. Further, it may be an artificial enzyme by gene recombination technique, partial hydrolysis or the like.
The form of glycosyltransferase is not particularly limited, and a dried product of enzyme protein, particles containing enzyme protein, liquid containing enzyme protein, and the like can be used.

The amount of glycosyltransferase used varies depending on the conditions of the glycosyltransferase reaction, the type of sugar, and the like. For example, in the case of cyclomaltodextrin glucanotransferase, 10 to 10,000 U / mL is preferable. Here, the activity is measured by the following method.
After reacting 100 μL of a solution containing 5 mM Tris-HCl buffer solution (pH 7.5), 0.05% (w / v) amylose and enzyme at 30 ° C. for 30 minutes, iodine solution (1 mg / mL KI, 0. The reaction is stopped by adding 2 mL (containing 1 mg / mL I ₂ , 3.8 mM HCl), and the absorbance at a wavelength of 660 nm is measured and quantified. The amount of enzyme that decreases the absorbance by 1% per minute is defined as 1 unit (U).

  In the step (3), it is possible to select the reaction temperature and the pH of the reaction solution as the conditions for the glycosyltransferase reaction in accordance with the characteristics of the enzyme to be used. For example, when cyclomaltodextrin glucanotransferase is used, pH 3 It is preferable to set it to -7, and also it is preferable to set it as 4-6.5.

  In addition, the reaction temperature is preferably 10 to 80 ° C., more preferably 20 to 60 ° C., and further preferably 35 to 55 ° C. from the viewpoint of the stability of glycosyltransferase and the improvement of the reaction rate of the glycosyltransferase reaction.

  Although the reaction time varies depending on the type of glycosyltransferase, etc., the water-insoluble polyphenols (A ′) from which rhamnose has been released are improved, the sugar adduct of the poorly water-soluble polyphenols from which the target rhamnose has been released is used. From the viewpoint of improving productivity, for example, 0.5 to 120 hours are preferable, 1 to 100 hours, and further 2 to 20 hours are preferable.

  According to the method of the present invention, a sugar adduct of poorly water-soluble polyphenols from which rhamnose is eliminated from poorly water-soluble polyphenols (A) having a rhamnoside structure in a yield of 40 to 100%, and further 45 to 95%. Can be manufactured. The conversion rate can be calculated by the formula (1) described in Examples below.

  The sugar adduct of poorly water-soluble polyphenols from which rhamnose has been obtained obtained by the method of the present invention is a compound in which at least one sugar is bound to the poorly water-soluble polyphenols (A ′) from which rhamnose has been eliminated. The type of sugar that binds to the poorly water-soluble polyphenols (A ′) from which rhamnose has been removed is not particularly limited, but is selected from 4 to 6 carbon sugars such as glucose, galactose, fructose, rhamnose, xylose, arabinose, and erythrose. At least one or more are preferred. In addition, the number of sugar bonds is preferably 1 to 10, more preferably 1 to 6, and the sugar binding site to the poorly water-soluble polyphenols from which rhamnose has been removed is a phenolic hydroxyl group or a glycoside. It is the sugar residue of the body. The binding mode between the poorly water-soluble polyphenols and these sugars may be either α-bond or β-bond.

  The dissolution amount of the poorly water-soluble polyphenols sugar adduct obtained by removing the rhamnose obtained by the method of the present invention is preferably 1 g / L or more, more preferably 2 g / L or more, still more preferably 5 g / L. L or more, more preferably 10 g / L or more. The amount dissolved in this specification is the amount dissolved in water at 25 ° C.

  The sugar adduct of polyphenols from which rhamnose is eliminated obtained by the production method of the present invention can be used for various foods and beverages, pharmaceuticals and the like. For example, as food and drink, confectionery such as beverages, breads, noodles, cookies, snacks, jelly, dairy products, frozen foods, instant foods such as powdered coffee, starch processed products, processed meat products, and other processed foods , Liquid, solid or semi-solid foods and beverages such as seasonings and dietary supplements. In particular, it is useful to be used for a packaged beverage. Examples of the packaged beverage include tea-based beverages such as green tea, and non-tea beverages such as sports beverages, isotonic beverages, and near water.

[Measurement of poorly water-soluble polyphenols]
Measurement of poorly water-soluble polyphenols was carried out by a gradient method using a high-performance liquid chromatograph manufactured by Hitachi, equipped with an intact column Cadenza CD-C18 (4.6 mmφ × 150 mm, 3 μm) and a column temperature of 40 ° C. . The mobile phase A solution was 0.05 mol / L acetic acid aqueous solution, the B solution was acetonitrile, and the solution was fed at 1.0 mL / min. The gradient conditions are as follows.
Time (min) A liquid (%) B liquid (%)
0 85 15
20 80 20
35 10 90
50 10 90
50.1 85 15
60 85 15
The sample injection amount was 10 μL, the detection was quantified by the absorbance of rutins at a wavelength of 360 nm, and hesperidins at a wavelength of 283 nm.

[Measurement of log P value of poorly water-soluble polyphenols]
It was measured according to the flask shaking method described in Japanese Industrial Standard Z7260-107. First, 1-octanol and distilled water were equilibrated by shaking at 25 ° C. for 24 hours. Next, 10 mg of polyphenol was weighed into a glass bottle with a lid, 4 mL each of 1-octanol and distilled water that had been equilibrated were added, and the mixture was shaken at 25 ° C. for 4 days. The 1-octanol phase and the aqueous phase were separated by centrifugation, and the concentration of polyphenols in each phase was measured by HPLC in the same manner as in the above [Measurement of poorly water-soluble polyphenol]. The value obtained by taking the common logarithm of the distribution coefficient between the two phases was defined as the logP value.

[Measurement of pH of aqueous medium and transglycosylation reaction solution]
The pH of the reaction solution after completion of the distilled water and rhamnose elimination reaction was measured at 25 ° C.

[Calculation of conversion rate of rhamnose elimination reaction]
The conversion rate in the step (2) was calculated by the following formula (1).
Conversion rate [%] = (Concentration of poorly water-soluble polyphenols (A ′) from which rhamnose was eliminated at the end of step (2)) / (Slightly water-soluble polyphenols having a rhamnoside structure at the start of step (2) (A ) Density) × 100 (1)

[Calculation of yield of polyphenol sugar adduct]
The yield of the sugar adduct of a poorly water-soluble polyphenol from which rhamnose was eliminated through the steps (2) and (3) was calculated by the following formula (2).
Yield [%] = (Concentration of sugar adduct of poorly water-soluble polyphenols from which rhamnose has been eliminated at the end of step (3)) / (Slightly water-soluble polyphenols having a rhamnoside structure at the start of step (2) (A ) Density) × 100 (2)

Example 1
Hesperidin preparation (Hesperidin “Hamali” (trade name), manufactured by Hamari Pharmaceutical Co., Ltd., hesperidin (HES) content 90%) is dispersed in distilled water (pH 6.5) at a concentration of 10 g / L, and slurry is supplied. Stirred uniformly in the tank. The liquid in the slurry supply tank was supplied at a rate of 100 mL / min to a stainless steel flow-type processing device (manufactured by Nitto Koatsu Co., Ltd.) having an internal volume of 100 mL, and heat treatment was performed at 150 ° C. (average residence time 1 minute). The pressure in the processor was adjusted to 0.6 MPa by an outlet valve. The heat treatment liquid was extracted from the outlet of the processor, cooled to 25 ° C. with a heat exchanger, passed through a sintered metal filter having a pore diameter of 7 μm, and then the pressure was returned to atmospheric pressure with an outlet valve to obtain a heat treatment liquid. The cooling rate obtained from the cooling time from 150 ° C. to 90 ° C. was 8 ° C./s.

  Five minutes after the end of the heat treatment, 10 mL of the obtained heat treatment solution was put into a glass bottle, and 0.005 g of hesperidinase (manufactured by Sigma-Aldrich, 3 units / g) was added. The hesperidin concentration in the reaction solution at the start of the rhamnose elimination reaction was 2.5 g / L, which was a supersaturated dissolution state significantly higher than the solubility in water at 25 ° C. (0.02 g / L). The reaction solution was shaken at 70 r / min on a rotary shaker at 50 ° C. for 7 hours. The enzyme was deactivated by immersing the glass bottle in boiling water for 1 minute to stop the reaction. The monoglucosyl aglycone concentration after completion of the elimination reaction was 2.4 g / L, and the yield was 0.024 g.

  Ten minutes after the termination of the elimination reaction, 0.2 g of γ-cyclodextrin and 0.5 mL of contizyme were added. The mass ratio of γ-cyclodextrin to monoglucosyl aglycone was 8.3. The sugar transfer reaction was performed by shaking at 70 r / min for 5 hours at 50 ° C. on a rotary shaker. The enzyme was deactivated by immersing the glass bottle in boiling water for 1 minute to stop the reaction.

Comparative Example 1
0.0279 g of the hesperidin preparation and 10 mL of distilled water were placed in a glass bottle. Although hesperidin was 2.5 g / L, it was in a suspended state. 0.005 g of hesperidinase was added, and rhamnose elimination reaction and sugar transfer reaction were carried out in the same manner as in Example 1.

Example 2
A heat treatment liquid was obtained in the same manner as in Example 1 except that the rutin preparation (manufactured by Tokiwa Plant Chemistry Laboratories, Inc., rutin content 100% by mass) was changed to 5 g / L instead of 10 g / L of the hesperidin preparation.
Five minutes after the end of the heat treatment, 10 mL of the obtained heat treatment solution was placed in a glass bottle, and 0.01 g of naringinase (manufactured by Amano Enzyme, Inc., trade name naringinase amano) was added. The rutin concentration in the reaction solution at the start of the rhamnose elimination reaction was 5.0 g / L, which was a supersaturated dissolution state significantly higher than the solubility in water at 25 ° C. (0.03 g / L). The reaction solution was shaken at 70 r / min on a rotary shaker at 50 ° C. for 7 hours. The enzyme was deactivated by immersing the glass bottle in boiling water for 1 minute to stop the reaction.
Ten minutes after the termination of the reaction, 0.2 g of γ-cyclodextrin and 0.5 mL of contizyme were added, and the sugar transfer reaction was carried out by shaking at 70 r / min with a rotary shaker at 50 ° C. for 5 hours. The enzyme was deactivated by immersing the glass bottle in boiling water for 1 minute to stop the reaction.

Comparative Example 2
0.05 g of rutin preparation and 10 mL of distilled water were put in a glass bottle. Rutin was in a suspended state at 5.0 g / L. 0.01 g of naringinase was added, and rhamnose elimination and transglycosylation were carried out in the same manner as in Example 2.

Example 3
A heat treatment liquid was obtained in the same manner as in Example 2 except that the heat treatment temperature was 120 ° C. and the pressure was 0.3 MPa. A rhamnose elimination reaction was carried out in the same manner as in Example 2. Although the rutin concentration in the reaction solution was 5.0 g / L, it was in a slightly suspended state.

Example 4
10 mL of the heat treatment liquid obtained in Example 2 was placed in a glass bottle and allowed to stand. A rhamnose elimination reaction was performed in the same manner as in Example 2 180 minutes after the end of the heat treatment. Although the rutin concentration in the reaction solution was 5.0 g / L, it was in a suspended state.

Comparative Example 3
10 mL of the heat treatment liquid obtained in Example 2 was placed in a glass bottle and allowed to stand. A rhamnose elimination reaction was performed in the same manner as in Example 2 360 minutes after the end of the heat treatment. Although the rutin concentration in the reaction solution was 5.0 g / L, it was in a suspended state.

  The results are shown in Table 1.

As is clear from Table 1, according to the method of the present invention, the amount of poorly water-soluble polyphenols having a rhamnoside structure can be remarkably increased, and the rhamnose elimination reaction proceeds efficiently, resulting in a high level. A sugar adduct of poorly water-soluble polyphenols was obtained at a conversion rate.
On the other hand, in Comparative Examples 1 and 2, since the amount of hesperidin and rutin dissolved at the start of the rhamnose elimination reaction was low and in a suspended state, the conversion rates of the rhamnose elimination reaction and the transfer reaction were low. In Comparative Example 3, rutin and hesperidin were dissolved immediately after the end of the heat treatment, but after 360 minutes from the heat treatment, the suspension was in a suspended state and the reaction rate thereafter was low.

Claims (6)

Next steps (1), (2) and (3):
(1) In the presence of an aqueous medium, the poorly water-soluble polyphenols (A) having a rhamnoside structure are related to the solubilizer (C).
0 ≦ (C) / ((A) + (C)) <0.1
The heat treatment is performed at 110 to 180 ° C. under the above conditions to obtain a heat treatment liquid, and then the cooling rate calculated from the time required for the heat treatment temperature to decrease to 90 ° C. is 3 ° C./s or more and 100 ° C./s under the following conditions, steps it cool the heat-treated solution from the heat treatment temperature to 90 ° C. or less,
(2) The rhamnose elimination reaction is started within 300 minutes after the heat treatment for the obtained heat-treated liquid after cooling to obtain a reaction liquid containing the poorly water-soluble polyphenols (A ′) from which rhamnose is eliminated. A step, and (3) adding a sugar donor (B) and a glycosyltransferase to the resulting reaction solution to obtain a sugar adduct of a poorly water-soluble polyphenol from which rhamnose has been eliminated by a glycosyltransferase reaction,
A process for producing a sugar adduct of a poorly water-soluble polyphenol.   The method for producing a sugar adduct of a poorly water-soluble polyphenol according to claim 1, wherein the pH of the aqueous medium used in the step (1) is 3 or more and less than 7.   The method for producing a sugar adduct of a poorly water-soluble polyphenol according to claim 1 or 2, wherein the log P value of the poorly water-soluble polyphenol (A) having a rhamnoside structure is -1.0 to 4.0.   The process for producing a sugar adduct of a poorly water-soluble polyphenol according to any one of claims 1 to 3, wherein in the step (3), the temperature of the sugar transfer reaction is 10 to 80 ° C.   Furthermore, the manufacturing method of the sugar addition product of the poorly water-soluble polyphenols of any one of Claims 1-4 including the process of removing solid from the heat processing liquid obtained at the process (1).   The mass ratio (B) / (A ') of the sugar donor (B) to the poorly water-soluble polyphenols (A') from which rhamnose is eliminated at the start of the transglycosylation reaction is 1-30. The manufacturing method of the sugar adduct of the poorly water-soluble polyphenols any one of -5.