JP3052172B2 - Method for producing tea catechins - 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 producing tea catechins, and more particularly, to a method for producing high-purity catechins at low cost using tea leaves as a raw material and removing caffeine and the like. It relates to a manufacturing method.

[0002]

2. Description of the Related Art Tea is one of the favorite drinks widely consumed around the world, and is botanically a woody evergreen tree of the genus Camellia of the family Camellia. Unfermented tea), oolong tea (semi-fermented tea), black tea (fermented tea), and the like, and these products can be obtained basically from the same tea plant.

[0003] Tea catechins contained in tea leaves are polyphenol compounds, such as (-) epicatechin and (-) epicatechin.
Epigallocatechin, (−) epicatechin gallate,
(-) Epigallocatechin gallate, (+) catechin,
(+) Gallocatechin and (+) gallocatechin gallate, which in a broad sense can include oligomers such as theaflavins condensed with these catechins (hereinafter referred to as “catechin oxidized polymer”), and tea leaves 9
-30% by weight.

[0004] Among these catechins, the main catechins in tea leaves are (-) epicatechin, (-) epigallocatechin, (-) epicatechin gallate, and (-) epigallocatechin gallate (hereinafter referred to as "-"). In recent years, studies on these catechins have confirmed chemical and pharmacological effects such as antioxidant, antibacterial, deodorant, blood cholesterol suppression, blood pressure lowering, and antitumor. It is expected to develop applications for food materials and functional foods.

[0005] The above-mentioned tea catechins which are the object of the present invention include (-) epicatechin, (-) epigallocatechin, (-) epicatechin gallate and (-), which are the main catechins in tea leaves. In addition to the four epigallocatechin gallates, (+) catechin, (+) gallocatechin and (+) gallocatechin gallate may be included.

The catechin oxidized polymer includes proanthocyanidins, assamicin, oolong homobisflavans, hydrolyzable tannins, theaflavins, theaflagalins, and theasinensins.

[0007] In order to produce such tea catechins with high purity, it is essential to separate impurities that coexist with the tea catechins in tea leaves. The impurities include caffeine,
There are saccharides, amino acids, proteins, organic acids, lipids, pigments, and catechin oxidized polymers.

Of these components, catechin oxidized polymers fall into the category of tea catechins in a broad sense, but have no effect such as antioxidation and have a brown color. May be problematic.

Therefore, it is desirable that the tea catechins to be produced in the present invention do not contain catechin oxidized polymers and that they be removed.

[0010] Other sugars, amino acids and the like are also desired to be removed from the tea catechins in order to exert their various actions sufficiently, because they serve as nutrient sources for common bacteria.

[0011] Conventional methods for producing tea catechins include:
Methods using liquid-liquid partitioned extraction are known.

For example, Yoshida (Tea Research Report No. 13, p. 4, April 1959) introduces a method of extracting green tea with water, removing caffeine with chloroform, and extracting with ethyl acetate.

Japanese Patent Publication No. 44234/1994 describes that a natural antioxidant can be industrially produced by a similar method.

Japanese Patent Application Laid-Open No. 64-9922 describes that tea extract can be used to remove caffeine with hexane and chloroform, and the target tea catechins can be extracted with ethyl acetate. .

[0015] In these methods, the extraction efficiency is poor due to the fact that the tea extract as the starting material contains a large amount of contaminants and the relationship of the extraction partition coefficient, and the tea extract is repeatedly extracted to obtain the desired tea catechins with high purity. Is considered necessary.

Further, since chloroform, which is a chlorine-based organic solvent, is used to remove caffeine, a safety problem is expected when it is used in foods and the like.

Japanese Patent Application Laid-Open No. 3-14572 discloses a catechin polymer obtained by extracting a plant such as tea with a lower alcohol aqueous solution as a raw material and using gel beads and a multistage batch process capable of high load conditions. Alternatively, it is described that tea catechins including a complex can be produced.

In this method, since the catechin polymer or the complex is contained in the product and the reaction tank is stirred or shaken during the production, the gel beads are expected to be worn.

On the other hand, the present inventors have used a tea extract obtained by extracting tea leaves with warm water as a raw material, filled a chromatographic column with an adsorbent, and selectively adsorbed tea catechins to the adsorbent to obtain a hydrophilic organic compound. A chromatography column adsorption / separation technology for producing high-purity tea catechins by eluting and recovering tea catechins with a solvent has been developed (JP-A-2-31474).
issue).

According to this method, the tea extract (water-soluble component) is injected into a chromatographic column filled with an adsorbent, and the elution is carried out sequentially by changing the concentrations of water and a hydrophilic organic solvent to thereby remove impurities first. Removed components remaining on the chromatographic column were replaced with methanol, ethanol, or acetone.
Alternatively, about 10 to 65 vol% of a mixture thereof
By eluting with an aqueous solution, tea catechins as target components can be recovered with high purity and a high recovery rate.

[0021] In particular, caffeine among the contaminants is not a problem during normal ingestion, but there are reports of disorders due to excessive ingestion and effects on neonates. Is required.

Caffeine is contained in tea leaves in an amount of about 2 to 5 wt%, depending on the type of tea, and when tea leaves are extracted with warm water or the like, the amount of tea extract becomes about 4 to 10 wt% in terms of fixed components in a tea extract.

When producing tea catechins using this tea extract, if caffeine is not removed, 10 to 2
It is about 5% by weight.

Infants who do not like caffeine,
In view of providing caffeine-removed tea catechins for foods used by pregnant women, persons with liver impairment and the like, and foods for livestock and pet animals sensitive to caffeine, the present inventors have proposed the above-mentioned Japanese Patent Application Laid-open No. The optimum conditions for removing caffeine were studied among the techniques developed in US Pat.

As a result of this examination, caffeine can be removed by optimizing the solvent concentration and the flow rate of the hydrophilic organic solvent used for washing contaminants according to the adsorbent used, without using chloroform or the like. It was confirmed that tea catechins could be produced.

However, in order to remove caffeine, a large amount of a low-concentration aqueous solution having a solvent concentration of about 5 to 15% by weight is required, and a new problem has arisen in that the cost for solvent recovery increases.

However, subsequent studies have revealed that the product thus produced contains a brown catechin oxidized polymer having no action such as antioxidation.

For this reason, first, a new separation / analysis method at an analytical level for grasping the behavior of a catechin oxidized polymer from a raw material to a final product is found, and then the catechin oxidized polymer is removed at an industrial level. As a result of studying the method, a method for producing tea catechins capable of efficiently removing a catechin oxidized polymer by organically combining a separation step by chromatographic separation and a liquid-liquid extraction step was developed (JP-A-Hei. No. 4-182479).

According to this method, for example, at least contaminants other than the catechin oxidized polymer are removed in advance in the separation step using a chromatographic column, so that a small amount of extraction solvent is used in the next liquid-liquid extraction step. The catechin oxidized polymer can be removed, and high-purity tea catechins can be produced.

By combining such a chromatographic separation step and a liquid-liquid extraction step, tea catechins can be produced more efficiently with higher purity and higher recovery than when these are used alone. The entire manufacturing process must be complicated, and the complicated manufacturing process is a new problem in manufacturing.

[0031]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and has as its object to produce tea catechins from tea as a raw material in a safe manner on a food hygiene scale on an industrial scale. The catechin oxidized polymer is removed without using a liquid-liquid extraction step, as well as the saccharides, impurities such as amino acids and caffeine contained therein,
An object of the present invention is to provide a method for producing high-purity tea catechins at low cost.

[0032]

That is, the present invention provides a method for producing high-purity tea catechins after efficiently and inexpensively removing caffeine and catechin oxidized polymer contained in tea leaves. Thus, in the present invention, as a result of various studies in view of the actual situation of solvent recovery generated from the step of removing water-soluble contaminants, a method for washing water-soluble contaminants without using a solvent was completed. Things.

That is, the first invention focuses on selecting a synthetic adsorbent to be packed in a chromatographic column and washing the chromatographic column with water without using an organic solvent. An extraction step of obtaining a tea extract by extraction, an adsorption step of injecting the tea catechin components into the chromatographic column by injecting the tea extract into a chromatographic column filled with a gel-type synthetic adsorbent, and water By washing the chromatographic column using a, a washing step to remove water-soluble impurities other than at least caffeine-containing tea catechins, and the components remaining in the chromatographic column, methanol, ethanol or acetone solvent, or An elution step of eluting a 50 to 100 vol% aqueous solution comprising these mixtures as an eluent, It is a method of manufacturing a tea catechins to be.

In the above-mentioned washing step, at least the catechin oxidized polymer can also be removed.

The above gel type adsorbent can be made of a vinyl polymer as a base material.

In the above-mentioned elution step, tea catechins having a solid content of 80% by weight or more in the recovered liquid from the chromatography column can be obtained.

In the above-mentioned elution step, tea catechins having a solid content of 90 wt% or more in the recovered liquid from the above-mentioned chromatographic column can be obtained.

By the washing operation in the washing step, 80% or more of caffeine and 80% or more of the catechin oxidized polymer can be removed.

The loading amount of the above-mentioned tea extract can be converted to a tea extract concentration of 15 Brix% to have a volume ratio of 0.5 to 6 with respect to the adsorbent.

The water in the washing step may have a volume ratio of 2 to 6 with the adsorbent.

The eluent may have a volume ratio of 2 to 6 with the adsorbent.

A hydrophilic vinyl polymer was used as the adsorbent, and the loading amount of the tea extract was converted to 15 Brix% to a volume ratio of 1 to the adsorbent, and a volume ratio of 4 to the adsorbent was 4
Of water and a volume ratio of 3
The solution can be passed through with an aqueous solution of ol% ethanol.

The above tea catechins include (-) epicatechin, (-) epigallocatechin, (-) epicatechin gallate, (-) epigallocatechin gallate, (+) catechin, (+) gallocatechin, and (+) ) Gallocatechin gallate.

Next, the second invention focuses on washing the chromatographic column on which caffeine and tea catechins are adsorbed with warm water, and obtains a tea extract by extracting water-soluble components from tea leaves. An extraction step, an adsorption step of injecting the tea extract into a chromatographic column filled with a synthetic adsorbent, thereby adsorbing the tea catechin components onto the chromatographic column, and washing the chromatographic column with water and hot water in sequence. Thus, a washing step for removing water-soluble contaminants other than at least caffeine-containing tea catechins, and the components remaining on the chromatographic column were replaced with 50 to 100 vol. Of a methanol, ethanol or acetone solvent, or a mixture thereof. % Elution using an aqueous solution as an eluent. .

The above synthetic adsorbent can be used as an adsorbent having styrene / divinylbenzene or methacrylate as a base.

The temperature range of the hot water is 60 to 90
° C.

The concentration of the aqueous solution consisting of one of the above-mentioned methanol, ethanol or acetone solvents, or a mixture thereof, can be 50 to 100%.

Hereinafter, the present invention will be described more specifically. According to the present invention, a tea extract containing, for example, a water-soluble component extracted from tea leaves with warm water is passed through a chromatographic column filled with an adsorbent, and contaminants including caffeine and catechin oxidized polymer are washed with water. A tea eluting step wherein the target tea catechins remaining on the chromatographic column are desorbed with a 50 to 100% aqueous solution of methanol, ethanol or acetone, or a mixture thereof to recover the tea catechins. This is a method for producing catechins.

FIG. 1 schematically shows the steps of the method for producing tea catechins according to the first invention.

The above-mentioned tea catechins of the above objects have a relatively large content and are the main components having various effects (-) epicatechin, (-) epigallocatechin, (-)
It is a mixture of epicatechin gallate and (−) epigallocatechin gallate, and may further contain (+) catechin, (+) gallocatechin and (+) gallocatechin gallate as minor components.

The adsorbent is desirably an adsorbent based on a hydrophilic vinyl polymer which is a gel filtration adsorbent.

This will be described more specifically. Tea, which is a raw material of the present invention, is used in various forms such as raw tea, unfermented tea, semi-fermented tea, and fermented tea. For example, by extracting with hot water of 80 ° C. or more for about 5 to 15 minutes, To obtain a tea extract of about 2 to 4 Brix% containing a water-soluble component.

The tea extract may be used by concentrating it as needed, or may be used without concentration.

When the tea extract is concentrated, it is concentrated to a maximum of about 15 Brix% in order to suppress the formation of a precipitate (cream down) due to the reaction between tea catechins and caffeine.

That is, about 2 to 15 Brix% is applied as the concentration of the tea extract used in the present invention.

It is to be noted that a tea extract prepared by drying a concentrate of the tea extract and then dissolving the solid powder in water again may be used.

When the concentration of the tea extract is high, that is, when the concentration of the tea catechins is high, the equilibrium adsorption amount is relatively large, and the processing amount per unit chromatographic column adsorbent increases, but the viscosity of the tea extract also increases. As a result, the chromatographic column pressure loss increases.

On the other hand, in the case of a tea extract having a low concentration without performing the concentration operation, the equilibrium adsorption amount is small, and the throughput per unit of the chromatographic column adsorbent is low. .

Therefore, the necessity of concentration of the tea extract should be appropriately determined in consideration of the supply system of the raw materials and restrictions on the design of the apparatus.

The tea extract is passed through a chromatography column filled with an adsorbent.

As a chromatographic column adsorbent, a gel type synthetic adsorbent such as a gel filtration type adsorbent having a vinyl polymer as a base, such as a hydrophilic vinyl polymer, can be used.

For example, there are Toyopearl HW40EC (manufactured by Tosoh Corporation) and Asahipak GS-520 (manufactured by Showa Denko).

In the case of an adsorbent based on styrene / divinylbenzene or methacrylic acid ester, tea catechins, caffeine and especially catechin oxidative polymerization under the same conditions as an adsorbent based on a hydrophilic vinyl polymer. It is difficult to separate objects.

When an adsorbent having a dextran derivative as a matrix can be used, tea catechins can be separated from caffeine and catechin oxidized polymers. These adsorbents are, for example, Sephadex LH-20.
(Pharmacia), etc., the particle size is small, so that a large pressure loss is caused when the liquid is passed through the column, and problems such as the compaction of the packed bed are expected.

The amount of the tea extract loaded on the chromatographic column was:
It is desirable to inject a tea extract at each concentration in advance and measure the amount until the tea catechins break through, and determine the measured value as a reference.

In determining the load, the load is set to 20 to 95% of the load up to the break point in consideration of slight movement of the adsorption zone of tea catechins in the next washing step with water.

When the load is less than 20%, the production efficiency per adsorbent decreases, and when the load is more than 95%, the tea flow of about 15% or more depends on the flow conditions such as the column shape and the flow rate. This results in loss of catechins.

Further, when the tea extract is loaded beyond the breakthrough point, the tea extract is no longer adsorbed by the adsorbent and flows out of the column, and the recovery rate of the tea catechins is significantly reduced.

The present inventors have conducted intensive studies on the loading amount of the tea extract, especially considering the separation characteristics between the oxidized polymer of caffeine and catechin and the target component tea catechins. It has been found that the volume ratio to the synthetic adsorbent should be 0.5 to 6 in terms of the solution concentration of 15 Brix%.

When the loading amount of the tea extract is represented by the weight of the water-soluble solids in the tea extract per unit adsorbent volume,
Desirably, it is 50 to 360 mg solids / ml-Resin.

The load of the tea extract was 360 mg solids / m
When l-Resin is exceeded, the adsorption band in the adsorption layer of the tea catechins becomes too long, and in the washing operation with water in the next step, the tea catechins pass through together with caffeine and the recovery rate decreases, or In a high-concentration tea extract, the chromatographic column pressure loss increases due to the influence of impurities.

When the loading amount of the tea extract is 50 mg solids / ml
When it is smaller than -Resin, it is possible to separate tea catechins and caffeine, but it is uneconomical because the production amount is simply reduced.

As described above, by passing the tea extract through the column, non-adsorbable impurities such as sugars and amino acids are discharged from the column outlet without being adsorbed by the adsorbent.

When a certain amount of the caffeine and catechin oxidized polymer is weakly adsorbed on the adsorbent, breakthrough starts and elutes from the column outlet.

The tea catechins, which are the target components, continue to be adsorbed to the adsorbent without breaking if the load amount is equal to or less than the set value of the load.

When the passage of the set amount of tea extract is completed, tea catechins and a part of caffeine and catechin oxidized polymer are adsorbed on the adsorbent in the chromatographic column, and the adsorbent in the adsorbent particles is inserted into the voids of the adsorbent particles. Contaminants remain.

Next, the chromatography column is washed with water. By this washing operation, non-adsorbable contaminants such as sugars and amino acids remaining in the sorbent space at the end of the tea extract injection are discharged.

Further, the caffeine and catechin oxidized polymer weakly adsorbed to the adsorbent are desorbed from the adsorbent and eluted from the column as the concentration equilibrium between the mobile phase and the stationary phase changes.

The amount of water used for washing at this time may be such that the volume ratio with the synthetic adsorbent is 2 to 6.

If the volume ratio is less than 2, the removal of caffeine and catechin oxidized polymer becomes insufficient, and if the volume ratio exceeds 6, the recovery of tea catechins due to partial breakthrough of the tea catechins decreases. .

In this washing step using water, the entire amount of tea catechins can be recovered in the next elution operation (elution step) without flowing out of the chromatographic column.

That is, the tea catechins remaining adsorbed on the column are desorbed using a 50-100 vol% aqueous solution of a hydrophilic organic solvent, for example, one of methanol, ethanol or acetone, or a mixture thereof as an eluent. Elute.

As a hydrophilic solvent to be used, a single solvent of ethanol is preferable in consideration of food addition and a solvent recovery treatment step.

If the solvent concentration is less than 50 vol%, the rate of elution of tea catechins is reduced, and the recovery rate of tea catechins is not preferred.

The amount of the eluent used in the elution step at this time may be such that the volume ratio with the synthetic adsorbent is 2 to 6.

When the volume ratio is less than 2, the recovery of tea catechins becomes insufficient, and when the volume ratio exceeds 6, the amount of tea catechins recovered by the eluent becomes very small.

The polarity of the mobile phase changes depending on these solvents,
Tea catechins easily desorb from the adsorbent and are discharged from the outlet of the chromatography column.

When the solvent concentration is less than 60 vol%, the risk of ignition and the like is reduced, which is convenient for production control. However, when the solvent concentration is less than 50 vol%, the dissolution rate becomes slow.

Therefore, the design of the solvent concentration may be appropriately determined in consideration of restrictions on the design of the apparatus.

When the amount of the solvent is less than 1.5 times the amount of the adsorbent based on the solvent, tea catechins are not completely eluted.

Finally, washing with water is performed to recover the solvent remaining in the chromatography column.

The tea catechins thus recovered are:
The product can be commercialized in the form of a concentrated liquid or a dry powder by performing conventional concentration and drying treatments.

Next, the second invention will be described. This invention, after injecting a tea warm water extract into a chromatographic column filled with a synthetic adsorbent, first wash the chromatographic column with water only,
Thereafter, caffeine is removed with hot water at 60 to 95 ° C., and the components remaining on the chromatographic column are eluted with an aqueous solution of about 50 to 100 vol% of one of methanol, ethanol or acetone or a mixture thereof. The gist is a method for producing such a class.

FIG. 2 schematically shows steps of a method for producing tea catechins according to the second invention.

After the tea extract has been passed through the chromatographic column, washing with water is performed first before washing with warm water to remove impurities solidified and denatured with warm water.

If the temperature of the hot water for washing is not higher than 60 ° C., the effect of removing caffeine cannot be sufficiently obtained.
Above ° C, a non-uniform liquid flow occurs due to the generation of bubbles in the chromatographic column.

First, impurities other than caffeine are separated from water-soluble components of tea leaves by washing with water by the synthetic adsorbent packed in the chromatographic column.

These contaminants have a property that they are hardly adsorbed by the adsorbent, and the contaminants remaining in the voids in the chromatographic column when the tea extract is passed through can be taken out of the chromatographic column by flushing with water. it can.

Caffeine has a relatively high adsorptive power to adsorbents among contaminants, and therefore has a low elution rate with water, but can increase the elution rate with warm water and can be taken out of the chromatographic column.

On the other hand, tea catechins have a strong adsorbing power to the adsorbent, do not elute with water or warm water, and use an aqueous solution of a hydrophilic organic solvent having a high solvent polarity as in the first invention. Can be desorbed and recovered.

[0101]

In the method for producing tea catechins according to the present invention, in the first invention, caffeine and catechin oxidized polymer are obtained, and in the second invention, high-purity tea catechins are obtained after removing caffeine efficiently. It can be manufactured at low cost.

Since water or hot water is used in the step of separating the caffeine and catechin oxidized polymer, the recovery of the organic solvent for eluting the caffeine and catechin oxidized polymer is unnecessary, and the production equipment Can also be simplified.

[0103]

Next, embodiments of the present invention will be described. However,
Before the explanation, a quantitative calculation method for tea catechins will be described.

In the chromatographic column separation operation, (1)
The amount (mass) of each component in the tea extract to be injected into the column,
(2) the amount of each component in the recovered liquid obtained in the elution step, (3)
The ratios of the respective components and (4) the weight of the solid content obtained by removing the solvent of the tea extract and the recovered liquid are defined as follows.

(1) Amount of each component in tea extract Ct (in): catechin Co (in): catechin oxidized polymer Cf (in): caffeine Ci (in): impurities (2) Recovery in elution step Amount of each component in liquid Ct (out): Tea catechins Co (out): Oxidized catechin polymer Cf (out): Caffeine Ci (out): Impurities (3) Ratio of each component = (recovery in elution step) Amount of each component in liquid)
/ (Amount of each component in tea extract) ηt: Ct (out) / Ct (in) ηo: Co (out) / Co (in) ηf: Cf (out) / Cf (in) ηi: Ci (out) / Ci (in) (4) Weight of solids in tea extract: W (in) W (in) = Ct (in) + Co (in) + Cf (in) + Ci (in). . . Formula: Weight of the solid content in the recovered liquid in the elution step: W (out) W (out) = Ct (out) + Co (out) + Cf (out) + Ci (out). . . formula

The purity of the tea catechins contained in the solid content in the eluate obtained by the chromatographic column separation operation can be represented by the following or formula. ηt · Ct (in) / (ηt · Ct (in) + ηo · Co (in) + ηf · Cf (in) + ηi · Ci (in)) × 100. . . Formula Ct (out) / W (out) × 100. . . formula

[0107] Tea catechins and caffeine can be quantitatively analyzed by high performance liquid chromatography (hereinafter, referred to as "HPLC") using, for example, standard samples under the analytical conditions shown in Table 1 in FIG.

Therefore, the catechin purity can be determined by the formula, and the recovery rate of tea catechins and caffeine can be calculated.

On the other hand, the catechin oxidized polymer and contaminants are composed of multiple components, and generally require a great deal of labor when analyzed by a conventional method.

Actually, in the early stage of the development, the present inventors selected mainly sugars and amino acids as representative components for the component analysis of contaminants, and measured the components by a conventional analysis method. It has only been confirmed that these representative components are removed.

[0111] From the knowledge of literature information and the like, it was expected that a small amount of catechin oxidized polymer was contained even if it contained catechin oxidized polymer. Was treated as.

However, in subsequent studies, it was also confirmed by gravimetric analysis and HPLC analysis that the product obtained by chromatographic column separation contained a considerable amount of catechin oxidized polymer.

Therefore, when tea catechins are industrially produced under optimal conditions, it is considered essential to quantitatively understand catechin oxidized polymers and contaminants. The behavior of oxidized polymer and contaminants was studied.

As a result, in the chromatographic column separation, the contaminants are adsorbed on an adsorbent, such as styrene / divinylbenzene, vinyl polymer, methacrylic acid ester, or dextran, which is packed in the applicable chromatographic column. In addition, it is possible to wash with water, and by optimizing washing conditions, the ratio of the component amounts of the contaminants = (the component amount in the recovered liquid in the elution step) / (the component amount in the tea extract) ηi : (Ci (ou
t) / Ci (in)) was confirmed to be ηi ≒ 0 by a gravimetric method.

FIG. 4 shows, as an example, a chromatographic column filled with adsorbent Sepabead SP850 (Mitsubishi Kasei) having styrene / divinylbenzene as a base, and a tea extract was passed through the chromatographic column and washed with water. It is a graph which shows the weight change of the contaminant measured at the column exit.

The catechin oxidized polymer can be analyzed relatively easily by the new HPLC analysis conditions studied by the present inventors (however, it is difficult to obtain a standard sample of the catechin oxidized polymer, As such, each component is not quantified.)

As an example, the HPLC analysis conditions are shown in Table 2 in FIG. FIG. 6 shows that an adsorbent HP1MG having a methacrylic acid ester as a base is packed in a chromatographic column,
It is a graph which shows the breakthrough curve of the component obtained by continuing flowing a tea extract to this.

The present inventors have already optimized caffeine and tea catechins by optimally setting the loading conditions of the tea extract in the adsorption step, the washing conditions in the washing step with water, and the eluent conditions in the elution step. When the tea extract was further passed through the chromatographic column beyond the optimal load condition, the tea catechins and caffeine and catechin oxidized polymer were almost the same as shown in FIG. Have been found to exhibit adsorption characteristics.

Although not shown in the figure, when the elution operation was performed with a 70 vol% aqueous acetone solution after the breakthrough adsorption operation, the tea catechins and caffeine and catechin oxidized polymer were almost the same. The characteristics were shown.

At this point, styrene / divinylbenzene or methacrylic acid ester, which is considered to be more economical in terms of the handling of the filler, the physical properties of the filler strength and the price, is used as the base material. Although the measurement was performed mainly using the adsorbents described above, tea catechins and catechin oxidized polymers have the same or similar basic chemical structures, and both have similar chromatographic column separation characteristics. Has been determined.

From the above results, when the test is performed under the chromatographic column separation conditions in which ηi = 0 and ηo = ηt, the above expression representing the purity of the tea catechins can be rewritten as an expression. ηt · Ct (in) / (ηt · (Ct (in) + Co (in)) + ηf · Cf (in)) × 100. . . formula

At this time, the parameters of the above formula and the formula can be easily quantified except for Co (in), and the formula and the formula are equal within the measurement error. Component amount of catechin oxidized polymer Co
(In) can be calculated, and these values are substituted into the above equation to determine the component amount C of the impurities in the tea extract.
It has become possible to obtain i (in).

Therefore, if the composition of the tea extract component is determined in advance by the above-described procedure, the analysis time can be significantly reduced thereafter.
Using data obtained under PLC conditions, the purity and the like of tea catechins can be analyzed by a formula.

The present inventors have continued to study chromatographic column separation, and have found that a method for removing catechin oxidized polymer (JP-A-2-313390) and a method for separating caffeine by washing with water (Japanese Patent Application No. Hei 4-31390). No. 113161).

In particular, it has been found that the latter can industrially produce tea catechins using an adsorbent containing a vinyl polymer as a base.

However, when analyzing the obtained measurement data, it was found that tea catechins and catechin oxidized polymers could not be converted to styrene / divinylbenzene or methacrylic acid ester even when an adsorbent having vinyl polymer as a base was used. It was carried out using an equation with a prejudice that it exhibits the same behavior as the adsorbent to be used.

However, the adsorption / elution characteristics of the adsorbent having a vinyl polymer as a base were surely grasped, for example, because excellent data exceeding the error range was obtained in the efficacy evaluation test of the obtained product. For this reason, the measurement was carried out under the HPLC conditions shown in FIG. 5 including the catechin oxidized polymer.

As a result, it was surprisingly discovered that tea catechins and catechin oxidized polymers were completely different from adsorbents based on vinyl polymers.

One example is shown in FIG. FIG. 7 is a graph showing a component breakthrough curve obtained by filling a chromatographic column with an adsorbent HW40EC having a vinyl polymer, particularly a hydrophilic vinyl polymer as a base material, as an adsorbent and continuing the passage of a tea extract.

As shown in the figure, the catechin oxidized polymer is closer to the adsorption behavior of caffeine than the tea catechins.
It was confirmed that tea breakthrough occurred before tea catechins.

As described above, it is difficult to obtain a standard sample covering all of the catechin oxidized polymer, and thus it is difficult to quantify the standard sample. However, the ratio of the integrated absorbance output of each peak, which is proportional to the concentration of each component, is used. Component ratio of catechin oxidized polymer (amount of each component in recovered liquid in elution step) /
(Amount of each component in tea extract) ηo: (Co (out) / C
o (in)) can be determined.

Therefore, by substituting ηo into the above equation, it becomes possible to analyze an adsorbent having a vinyl polymer as a base.

Accordingly, the previous examples in Japanese Patent Application No. 4-113161 were reexamined by changing the analysis data. The results are shown below together with these examples.

Examples 1, 2, 3, 4, Comparative Examples 1 and 2 and Reference Example 1 described below correspond to the first invention, and Example 5 and Comparative Example 3 correspond to the second invention.

[Example 1] A tea extract was prepared by extracting 1 kg of third tea with 15 kg of hot water at 95 ° C for 10 minutes, followed by filtration.
This was prepared by vacuum concentration and centrifugation.

A commercially available Toyopearl HW40 (trade name, manufactured by Tosoh Corporation) having a hydrophilic vinyl polymer as a base material was used as an adsorbent.
Liter, packed in a chromatographic column, and the tea extract 1
3 liters of 2 Brix%, 5 liters of water, and 7
2.5 liters of a 0 vol% ethanol aqueous solution were sequentially passed.

As a result, the ethanol recovery liquid was concentrated under reduced pressure,
Lyophilization gave 143 g of a solid powder.

As a result of the analysis, it was estimated that the purity of the target tea catechins was 72% by weight, caffeine was 0.9% by weight, and the remainder was a catechin oxidized polymer. The recovery rate of tea catechins was 95%.

Here, the amounts of tea catechins and caffeine were calculated by high performance liquid chromatography (HPLC) by dividing the value quantified by an absolute calibration curve method with a sample by the amount of solid powder.

FIG. 8 shows a chromatogram of HPLC. Table 3 in FIG. 9 shows the HPLC conditions.

The recovery was calculated by dividing the amount of tea catechins contained in the solid powder by the amount of tea catechins contained in the tea extract.

When the analysis is performed again according to the above formulas and the like, this example shows that the target catechin purity is actually 9%.
About 7.6% by weight, and the contents of caffeine and catechin oxidized polymer are 1.2% by weight and 1.1%, respectively.
It is estimated that it was about wt%. The product weight estimated from the analysis data is estimated to be about 105 g.

Example 2 The method of preparing a tea extract and the adsorbent are the same as in Example 1.

In the chromatographic column, the tea extract 8 Brix%
, 3 liters of water, and 3 liters of a 50 vol% methanol aqueous solution.

As a result, 97 g of a solid powder was obtained. The purity of tea catechins is 73wt%, caffeine 0.2wt%
The rest was a catechin oxidized polymer. The recovery rate of tea catechins was 98%.

According to another analysis according to the above formulas and the like, this example actually shows that the target catechin purity is about 99.5 wt%, and that the contents of caffeine and catechin oxidized polymer are 0.3 wt% and 0.3 wt%, respectively. 0.
It is estimated that it was about 2 wt%. The product weight estimated from the analysis data is estimated to be about 71 g.

[Comparative Example 1] The method of preparing a tea extract and the adsorbent are the same as in Example 1.

As an adsorbent, 1 liter of commercially available S-876 (trade name, manufactured by Duolite Co.) having styrene / divinylbenzene as a base was packed in a chromatographic column, and 3 liters of 8 Brix% of tea extract and 10 vol% of methanol were added thereto. 2 liters of an aqueous solution and 3 liters of a 50 vol% methanol aqueous solution were sequentially passed.

As a result, 94 g of a solid powder was obtained. The purity of tea catechins is 63wt%, caffeine 13wt%,
The rest was presumed to be catechin oxidized polymer. The recovery rate of tea catechins was 95%.

Under the present separation conditions, that is, 2 liters of a 10 vol% methanol aqueous solution, it is determined that the amount is not sufficient to desorb caffeine from the adsorbent. For this reason, only 13% of the amount of caffeine contained in the tea extract could be removed.

[Reference Example 1] The method of preparing a tea extract is the same as in Example 1.

As the adsorbent, commercially available synthetic adsorbent having methacrylic acid ester as a base, HP1 manufactured by Mitsubishi Kasei Co., Ltd.
Packing 1 liter of MG ™ into a chromatographic column,
3 liters of 10 Brix% of tea extract, 10 vol.
6 liter of 50% methanol aqueous solution and 50 vol%
3 liters of an aqueous methanol solution were sequentially passed.

As a result, 98 g of a solid powder was obtained. Tea catechins have a purity of 71 wt% and caffeine 1.3 wt%
The rest was presumed to be catechin oxidized polymer. The recovery rate of tea catechins was 97%.

Similar results were obtained when acetone was used instead of methanol as the solvent, but the amount of solvent used could be reduced by 5% compared to methanol.

Similar results were obtained when the adsorbent was replaced with Duolite S-876 (trademark) of Comparative Example instead of HP1MG (trademark).

It was confirmed that under the present separation conditions, that is, when 6 liters of a 10 vol% methanol aqueous solution was used, caffeine was desorbed even from an adsorbent having methacrylate or styrene / divinylbenzene as a base.

However, as compared with the comparative example, 10 times of 10 times
It is necessary to recover the solvent from 6 liters of an ol% methanol aqueous solution.

As described above, it was confirmed that caffeine could be removed with water at a high efficiency of 95 to 99% using a gel filtration adsorbent containing hydrophilic polyvinyl alcohol as a matrix.

The catechin oxidized polymer exhibits the same behavior as the tea catechins in the adsorbent based on styrene / divinylbenzene as described in the description of the present invention.
In Comparative Example 1 and Reference Example 1 shown here, it is assumed that the conventional analysis method can be used.

As described above, it was confirmed that caffeine could be removed with water at a high efficiency of 95 to 99% using a gel filtration adsorbent containing hydrophilic polyvinyl alcohol as a matrix.

The caffeine content in the thus-obtained tea catechins was less than 1% by weight, which was 0.01 to 0.1 times as much as that without removing it.

It was confirmed that the adsorbent having styrene / divinylbenzene or methacrylic acid ester as a base can remove caffeine as in Comparative Example 1 by optimizing the conditions. It was difficult to remove caffeine only by washing with water as in the case of using a polyvinyl alcohol adsorbent.

Example 3 1.4 kg of green tea was extracted with 21 kg of hot water at 80 ° C. for 15 minutes, and the extract was filtered and centrifuged to remove insoluble solids.
2.2 liters of x% tea extract was obtained.

Two liters of this tea extract was injected into a chromatography column packed with 1 liter of Toyopearl HW40EC (trademark). Next, it was washed with 3 liters of water.

Lastly, 4 liters of a 60 vol% ethanol aqueous solution was injected to obtain a recovered liquid.

Further, the raw material tea extract, the water-washed fraction and the ethanol aqueous solution recovered fraction were analyzed by high performance liquid chromatography (HPLC).

The analysis conditions of the high performance liquid chromatography are shown in Table 4 in FIG. The chromatograms of the high performance liquid chromatography are shown in FIG.
1, FIG. 12 and FIG.

Further, the amounts of each component of the raw material tea extract and the ethanol aqueous solution recovered fraction were measured by quantitative analysis by high performance liquid chromatography.

As a result of analysis according to the above formulas and others,
(-) Epicatechin, (-) epigallocatechin, (-)
The total recovery of epicatechin gallate and (−) epigallocatechin gallate catechin was 94.2%, and the purity of catechin was 9
It was 7.3 wt%.

The contents of caffeine and catechin oxidized polymer were 2.0 wt% and 0.7 wt%, respectively.
Met.

The product weight estimated from the HPLC measurement result was 58.8 g.

When the solid content of a part of the ethanol aqueous solution recovered fraction was measured, it was 62 g in terms of the total amount of the product.
Met.

The catechin purity determined by the above equation using the solid content data was 92.2% by weight.

The product weight differs by 5.2% depending on the measurement method, and a measurement error is considered. However, it is presumed that other product components such as (+) catechin, (+) gallocatechin and (+) gallocatechin gallate are present.

Example 4 2.2 kg of green tea was extracted with 35 kg of hot water at 80 ° C. for 15 minutes, and the extract was filtered and centrifuged to remove insoluble solids.
4.4 liters of x% tea extract was obtained.

The tea extract 4 was added to a chromatographic column packed with 1 liter of Asahipak GS-520 (trademark).
One liter was injected. Next, it was washed with 3 liters of water.

Finally, 3 liters of a 70 vol% ethanol aqueous solution was injected to obtain a recovered liquid.

In the same manner as in Example 3, the amounts of each component were measured for the tea extract and the aqueous ethanol recovered fraction as raw materials, and as a result, (-) epicatechin, (-) epigallocatechin, and (-) epicatechin gallate were obtained. , (-) Epigallocatechin gallate catechin, the total recovery was 90.5%, and the catechin purity was 96.4 wt%.

The contents of caffeine and catechin oxidized polymer were 2.7 wt% and 1.2 wt%, respectively.
Met.

The product weight estimated from the result of HPLC measurement was 90.0 g.

The solid content of a part of the recovered ethanol aqueous solution was measured.
It was 4 g.

The catechin purity determined by the above equation using the solid content data was 87.8% by weight.

[Comparative Example 2] A tea extract 2 obtained in the same manner as in Example 3 was placed in a chromatographic column packed with 1 liter of a methacrylic acid ester-based adsorbent HP1MG (manufactured by Mitsubishi Chemical).
One liter was injected. Next, it was washed with 1.7 liters of water.

Finally, 4 liters of a 60 vol% ethanol aqueous solution was injected to obtain a recovered liquid.

In the same manner as in Example 3, the amounts of each component were measured for the tea extract and the aqueous ethanol recovered fraction as raw materials. The results were as follows: (-) epicatechin, (-) epigallocatechin, (-) epicatechin gallate, The total recovery of (-) epigallocatechin gallate catechin was 81.2%, and the catechin purity was 59.2 wt%.

The contents of caffeine and catechin oxidized polymer were 19.1 wt% and 21.7 w%, respectively.
t%.

The product weight estimated from the HPLC measurement result was 83.3 g.

When the solid content of a part of the recovered fraction of the aqueous ethanol solution was measured, it was 94.4 g in terms of the total amount of the product.

The catechin purity determined by the above equation using the solid content data was 52.2% by weight.

[0190] Insoluble matter was found in the fraction recovered from the aqueous ethanol solution, but it was considered that some of the impurities were insolubilized and precipitated by the action of ethanol because the amount of water in the washing step was small.

As in each of the above-described embodiments, when an adsorbent having a hydrophilic vinyl polymer as a base is used, impurities including caffeine and catechin oxidized polymer can be removed with high efficiency only by washing with water. Tea catechins were obtained.

On the other hand, although some contaminants such as sugars and amino acids could be removed by the adsorbent having methacrylic acid ester as a matrix, it was difficult to separate tea catechins from caffeine and catechin oxidized polymer. there were.

Example 5 1 kg of tea was extracted with 10 liters of hot water for 10 minutes, and the juice obtained by pressing was collected, concentrated under reduced pressure, and centrifuged to obtain a tea extract (tea extract). 5
Liters were made.

In the chromatography column, a commercially available synthetic adsorbent HP1M having methacrylic acid ester as a base is used as an adsorbent.
What filled G 1.5 liter was used.

After injecting the whole amount of tea extract, distilled water 1.
The mixture was washed with 5 liters and 3.5 liters of warm water at 90 ° C., and the residual components were recovered with 3 liters of an 80 vol% methanol aqueous solution.

The crude tea catechins were produced in an amount of 112 g, had a purity of 85 wt%, and contained 5 wt% of caffeine.

Further, 92% of tea catechins were recovered with respect to the tea extract.

Comparative Example 3 The same tea extract and chromatographic column as in Example 5 were used.

After the whole amount of the tea extract was injected, washing and recovery were performed using 4.5 liters of a 15 vol% methanol aqueous solution and 3 liters of an 80 vol% methanol aqueous solution.

The crude tea catechins were produced in an amount of 121 g, and had a purity of 79 wt% and contained 12 wt% of caffeine.

Further, 92% of tea catechins were recovered with respect to the tea extract.

In Comparative Example 3, when 4.5 liters of a 40 vol. Methanol aqueous solution was used instead of 4.5 liters of a 15 vol% aqueous methanol solution, 18 wt% of caffeine was contained in crude catechins, and The effect was small.

The production amount of the crude tea catechins in Example 5 described above is 112 g, which is smaller than the production amount of 121 g in Comparative Example 3, because caffeine is not contained in the former case. It was confirmed that, in addition to the economical effect that the recovery of the low-concentration solvent was unnecessary, the removal of caffeine was more excellent.

[0204]

According to the present invention as described above, according to the present invention, contaminants contained in the tea leaves were removed with water or warm water, with further ethanol <br/> over food hygiene safe solvents such as Le, high recovery High-purity tea catechins can be produced on an industrial scale.

In particular, caffeine and catechin oxidized polymer, which have been pointed out as having problems with food hygiene safety and separation technology in connection with the removal operation in the prior art, were analyzed using only a chromatographic column separation operation. It can be economically removed.

Further, since impurities in the tea extract can be removed only with water or warm water without using a conventionally required low-concentration eluent, a solvent recovery facility for the low-concentration eluate is unnecessary. And a cheaper product can be provided.

[0207]

[Brief description of the drawings]

FIG. 1 is an explanatory view schematically showing steps of a method for producing tea catechins according to the first invention.

FIG. 2 is an explanatory view schematically showing steps of a method for producing tea catechins according to the second invention.

FIG. 3 is a chart 1 showing analysis conditions of high performance liquid chromatography (HPLC) in a method for quantitatively calculating tea catechins.

[Fig. 4] Fig. 4 shows a separation column filled with adsorbent Sepabead SP850 (Mitsubishi Kasei) having styrene / divinylbenzene as a base, and a tea extract solution was passed through the chromatography column and washed at the outlet of the chromatography column. It is a graph which shows the weight change of an object.

FIG. 5 is a chart 2 of analysis conditions of high performance liquid chromatography (HPLC) in a method for quantitatively calculating a catechin oxidized polymer. (However, it is difficult to obtain a standard sample of a catechin oxidized polymer, so that its components are not quantified.)

FIG. 6: Adsorbent H based on methacrylic acid ester
It is a graph which shows the breakthrough curve of the component obtained by packing P1MG into a chromatographic column and continuing to pass a tea extract through it.

FIG. 7 shows a component breakthrough curve obtained by packing a chromatographic column with an adsorbent HW40EC having a vinyl polymer as a base material, particularly a hydrophilic vinyl polymer as a base material, and continuously passing tea extract through the column. It is a graph relevant to the invention.

FIG. 8 is a diagram showing a chromatogram of high performance liquid chromatography according to Example 1 of the first invention.

FIG. 9: High performance liquid chromatography (HPLC)
4 is a chart 3 of the analysis conditions.

FIG. 10 is a chart 4 showing analysis conditions of high performance liquid chromatography in Example 3 of the first invention.

FIG. 11 is a view showing a chromatogram of high performance liquid chromatography as an analysis result.

FIG. 12 is a view showing a chromatogram of high performance liquid chromatography as an analysis result.

FIG. 13 is a view showing a chromatogram of high performance liquid chromatography as an analysis result.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiroshi Shinya 63-30 Yuyugaoka, Hiratsuka-shi, Kanagawa Prefecture Sumitomo Heavy Industries, Ltd. Inside Hiratsuka Research Institute (72) Inventor Fumihisa Ranabe 21 Goddess Sagara-cho, Haibara-gun, Shizuoka Prefecture Shares Inside the ITO EN Central Research Institute (72) Inventor Chuichi Takeo 21 Goddess of Sagara-cho, Haibara-gun, Shizuoka Prefecture Inside the ITO EN Central Research Institute (56) References JP-A 1-175978 (JP, A) JP-A-1- 299224 (JP, A) JP-A-2-311474 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 311/62

Claims (12)

(57) [Claims] 1. An extraction step of obtaining a tea extract by extracting a water-soluble component from tea leaves, and injecting the tea extract into a chromatographic column filled with a gel-type synthetic adsorbent, whereby tea is added to the chromatographic column. an adsorption step of adsorbing the catechins components, a washing step in the wash the chromatographic column with water, the remaining components to the chromatographic column, consisting of methanol, ethanol or one of acetone solvent or mixtures thereof, 50 and elution eluting the ～100Vol% aqueous solution as an eluent, and has a load amount of the tea extract in the adsorption step, this
The above gel-type synthesis in terms of tea extract concentration of 15 Brix%
The volume ratio with the adsorbent is set to 0.5 to 6, and in the washing step,
The volume of the water in the gel-type synthetic adsorbent is
By setting the ratio to 2 to 6, at least caffeine and
Water solubility other than tea catechins including catechin oxidized polymer
A method for producing tea catechins excluding contaminants . 2. The method for producing tea catechins according to claim 1, wherein the gel type adsorbent is made of a vinyl polymer as a base. 3. The method for producing tea catechins according to claim 1, wherein tea catechins having a solid content of 80 wt% or more in the recovered liquid from the chromatography column in the elution step are obtained. 4. The method for producing tea catechins according to claim 1, wherein tea catechins having a solid content of 90% by weight or more in the recovered liquid from the chromatography column in the elution step are obtained. 5. The method for producing tea catechins according to claim 1, wherein 80% or more of caffeine and catechin oxidized polymer are removed by a water washing operation in the washing step. 6. The method for producing tea catechins according to claim 1, wherein the eluent has a volume ratio of 2 to 6 with respect to the adsorbent. 7. A hydrophilic vinyl polymer is used as the adsorbent, and the amount of the tea extract is set to 15 Brix% in terms of a volume ratio of 1 to the adsorbent, and water having a volume ratio of 4 to the adsorbent is used. And then, the volume ratio with the adsorbent is 3 to 5
The method for producing tea catechins according to claim 1, wherein the liquid is passed through with a 5 vol% ethanol aqueous solution. 8. The tea catechins include (−) epicatechin, (−) epigallocatechin, (−) epicatechin gallate, (−) epigallocatechin gallate, (+) catechin, (+) gallocatechin, and 2. The method for producing tea catechins according to claim 1, wherein (+) gallocatechin gallate. 9. An extraction step of obtaining a tea extract by extracting a water-soluble component from tea leaves, and injecting the tea extract into a chromatographic column filled with a synthetic adsorbent, whereby tea catechins are added to the chromatographic column. An adsorption step for adsorbing the components, a washing step for removing water-soluble contaminants other than tea catechins containing at least caffeine by washing the chromatographic column with water and hot water sequentially, Eluted with an aqueous solution of 50 to 100 vol% of one of methanol, ethanol or acetone solvents, or a mixture thereof, as an eluent. 10. The method for producing tea catechins according to claim 9, wherein the synthetic adsorbent is an adsorbent having styrene / divinylbenzene or methacrylate as a base. 11. The temperature range of the hot water is set to 60
The method for producing tea catechins according to claim 9, wherein the temperature is set to 90C. 12. The method for producing tea catechins according to claim 9, wherein the concentration of the aqueous solution comprising one of the methanol, ethanol and acetone solvents or a mixture thereof is 50 to 100%. .