WO2001059143A1 - Method for producing monoglycosidated flavonoids - Google Patents

Abstract

The invention relates to a method for producing monoglycosidated flavonoids by enzymatic hydrolysis of rutinosides, using an enzyme immobilized on a carrier for the enzymatic hydrolysis. The inventive method reduces the costs for the enzymes, and simultaneously provides for a high degree of automation associated and an optimized space/time yield.

Description

 Process for the preparation of monoglycosidated flavonoids

The present invention relates to a process for the preparation of monoglycosidated flavonoids by enzymatic hydrolysis of rutinosides. The rhamno residue of the rutinosides is cleaved off enzymatically.

In the context of the present invention, rutinosides are compounds which contain a sugar-free component in which a radical of the formula (I)

is bound via a glycosidic bond. For example, the rutinosides are flavonoids with the bisgylcosidic unit shown in formula (I). Rhamnose and / or the corresponding glucopyranosides can be obtained from the rutinosides. The glucopyranosides are derived from the rutinosides in that instead of the residue of the formula (I) they contain a residue of the formula (I *)

included, which is bound to the sugar-free ingredient. For example, both rhamnose and isoquercetin can be obtained from rutin. Rhamnose is a monosaccharide, which is widespread in nature, but mostly occurs only in small amounts. An important source for rhamnose are, for example, the glycosidic residues of natural flavonoids, such as rutin, from which the rhamnose can be obtained by glycoside cleavage. Rhamnose, for example, plays an important role as a raw material for the representation of artificial flavors, such as furaneol.

Isoquercetin is a monoglycosidated flavonoid of the following structural formula (II)

As flavonoids (lat. Flavu = yellow), which are widely used dyes in plants, e.g. Glycosides of flavones which share the basic structure of flavone (2-phenyl-4H-1-benzopyran-4-one).

The sugar-free component of the flavonoids is the so-called agiykon. For example, isoquercetin is a glycoside of the aglycon quercetin (2- (3,4-dihydrophenyl) -3,5,7-trihydroxy-4H-1-benzopyran-4-one), which differs from flavone in that it contains five hydroxyl groups. In isoquercetin, the carbohydrate residue glucose is bound to the hydroxyl group in position 3 of quercetin. For example, isoquercetin is used as quercetin-3-O-ß-D-glucopyranoside or 2- (3,4-dihydroxyphenyl) -3- (ß-D-glucopyranosyloxy) -5,7-dihydroxy-4H-1-benzopyran-4- called on. However, it is also known, for example, under the name hirsutrin. Flavonoids or flavonoid mixtures are used, for example, in the food and cosmetics industry and are becoming increasingly important there. Especially monoglycosidated flavonoids, such as isoquercetin, are characterized by their good absorption capacity in the human body.

An example of a naturally occurring flavonoid with a bisglycosidic unit is rutin, which has the following structural formula (III):

Like isoquercetin, rutin is also a glycoside of the aglycone quercetin, the carbohydrate residue rutinose being bound to the hydroxyl group in position 3 of the quercetin. The carbohydrate residue in rutin consists of a glucose unit linked in the 1- and 6-position and a terminally bound rhamnose or 6-deoxymannose unit. Rutin is used, for example, as quercetin-3-O-ß-D-rutinoside or 2- (3,4-dihydroxyphenyl) -3- {[6-O- (6-deoxy-α-mannopyranosol) -ß-D-glucopyranosyl] oxy! ^> -5,7-dihydroxy-4H-1-benzopyran-4-one. However, it is also known, for example, under the names sophorin, birutan, rutabion, taurutin, phytomelin, melin or rutoside.

With three molecules of water of crystallization, rutin forms pale yellow to greenish needles. Anhydrous rutin has the property of a weak acid, turns brown at 125 ° C and decomposes at 214-215 ° C. Rutin, which is found in many plant species - often as a companion of vitamin C - occurs, for example, in citrus, in yellow pansies, forsythia, acacia, various Solanum and Nicotiana species, capers, linden flowers, St. John's wort, tea, etc., was isolated in 1842 from the garden rhombus (Ruta graveolens). Rutin can also be obtained from the leaves of buckwheat and the East Asian coloring drug Wei-Fa (Sophora japonica, Fabaceae), which contains 13-27% rutin.

It is desirable to produce both rhamnose and monoglycosidated flavonoid from natural raw materials, for example from flavonoids with a bisglycosidic unit. In this context, e.g. the splitting of rutinosides into rhamnose and the corresponding glucopyranosides is interesting.

Enzymatically catalyzed representations of rhamnose are described in the literature. For example, EP-A-0317033 describes a process for the preparation of L-rhamnose, the rhamnosidic binding of glycosides which keep rhamnose bound in the terminal position is achieved by enzymatic hydrolysis. In this cleavage, the substrate is usually carried out as a suspension in an aqueous medium. However, these reactions are mostly not very selective. For example, due to the bisglycosidic structure of the carbohydrate residue in rutin, a mixture of the two monosaccharides glucose and rhamnose is often formed. In addition, there is usually a high proportion of Agiykon quercetin and other undesirable by-products.

Furthermore, enzymatically catalyzed cleavages of rutin are also described in JP-A-01213293. However, reactions of this type carried out in aqueous media are usually also not very selective.

These methods described above use the enzyme in solution, ie native. The enzyme is added directly to the reaction solution. Although these processes can be carried out on a laboratory scale, they are not practical for industrial use because the enzyme cannot be recovered from the reaction solution for reuse. However, the one-time use of these expensive enzymes is not economical in a technical application. It is known that enzymes can be used industrially by binding them to a carrier. This process is referred to as "immobilization". The term "immobilized enzymes" is used by the European Federation of Biotechnology (1983) to summarize all enzymes "... that are in a state that permits their reuse" (Helmut Uhlig, Technical enzymes and their application, Carl Hanser Verlag, Munich Vienna 1991, p. 198). Despite this advantage, immobilization is, however, not suitable for all enzyme processes and has so far only been used to a limited extent. In particular, only two immobilized enzymes are used on an industrial scale: glucose isomerization with immobilized glucose isomerase and peniciliin G cleavage with immobilized penicillin amidase. Often immobilized enzyme processes cannot compete with free enzymes or chemical processes. The enzymes or the reaction conditions are often also unsuitable for immobilization. So there is no universal method for immobilization, and each enzyme must be viewed individually.

For example, in aqueous systems, which are important for the functionality of enzymes, there are solubility problems with rutinosides, which serve as a substrate in the enzymatic hydrolysis. This reaction is therefore preferably carried out with a supersaturated substrate solution, i.e. as a rutinoside suspension. In the case of a supersaturated solution in which the substrate is present as a solid, however, immobilization techniques cannot be used. There is a lack of selectivity between raw material and product particles and solid-bound enzyme.

It is therefore the object of the present invention to provide a process for the production of monoglycosidated enzymes which can be used on an industrial scale, high enzyme costs being avoided and, at the same time, a high degree of automation combined with a high space / time yield and high productivity and selectivity is achieved. This object is achieved by a process for the production of monoglycosidated flavonoids by enzymatic hydrolysis of rutionosides, in which an enzyme which is immobilized on a support is used for the enzymatic hydrolysis. Surprisingly, it was found that, despite the low solubility of rutinosides, enzymatic hydrolysis with an immobilized enzyme is possible. Due to the immobilization, the process can be carried out continuously or discontinuously and with a high effectiveness in comparison with the reaction with the native enzyme. The process according to the invention is characterized in particular by the fact that high automation of the entire process, including the recycling of the solvent and the monitoring of the enzyme activity, is possible.

Figure 1 shows the continuous production of isoquercetin from rutin as an example of the process according to the invention.

Suitable rutinosides for the process according to the invention are those which contain a 2-phenyl-4H-1-benzopyran-4-one base body as a sugar-free constituent or agiycon, which bears a radical of the formula (I) in position 3 and whose phenyl group pen, apart from position 3, can also be substituted one or more times by -OH or -0 (CH ₂ ) _n -H, where n is 1 to 8, n is preferably 1.

The substitution of the 2-phenyl-4H-1-benzopyran-one base body by -OH and / or -0 (CH ₂ ) _n -H preferably occurs in positions 5, 7, 3 'and / or 4'.

Rutinosides, which are represented by the formula (A), are particularly preferably used:

wherein RH, OH or OCH represents ₃ .

The compound in which RH represents is referred to as fighter olrutinoside; the rutinoside, in which R represents OCH ₃ , is known as isorhamnetin rutinoside. The compound in which R is OH is called rutin. Accordingly, rhamnose and fighter olglucoside can be obtained from fighter olrutinoside, rhamnose and isoquercetin from rutin rutinoside and rhamnose and isorhamnetin glycoside from isorhamnetin rutinoside.

The rutinoside rutin is particularly preferably used.

The rutinosides can be used in pure form or as mixtures of rutinosides as starting material for the process according to the invention. The rutinosides can also be contaminated with other flavonoids or with residues from rutinoside production without the reaction being adversely affected.

Usual hydrolases which are able to split off the rhamnose residue of the rutinosides can be used as enzymes for the enzymatic hydrolysis of the rutinosides. Hydrolases originating from the strain Penicillium decumbens are preferred won, used. Α-L-Rhamnosidases are particularly preferably used as enzymes, since they have a high selectivity for the hydrolysis of the rhamnose residue. Suitable α-L-rhamnosidases are, for example, hesperidinase, naringinase, and those described in Kurosawa et al. (1973), J. Biochem., Vol. 73: 31-37. It is particularly preferred to use the enzyme hesperidinase.

Both the rutinosides and the enzymes for the process according to the invention can be purchased as commercial products. It is also possible to obtain or produce the starting materials and enzymes by generally known methods.

The enzyme is immobilized on a suitable carrier. Typical carriers, such as silica gel, for example commercially available spherical or commercially available broken silica gels, for example Lichrosorb ^® , Lichroprep ^® , Lichrospher ^® and Trisoperl ^® , and commercially available polymeric carriers, for example Eupergit ^® , Fractogel ^® , in particular Fractogel epoxy ^® , and Fractoprep ^® , are used. Silica gel is the preferred carrier material.

Alternatively, magnetic particles can also be used as carriers. These are preferably carrier materials with a magnetic core. This core is usually coated with an inorganic oxide. The inorganic oxide is preferably silica gel. Examples of such magnetic carriers include MagneSil ™ (Promega Corp., Madison, Wisconsin, US), MagPrep ™ (Merck) and AGOWAmag ™ (AGOWA GmbH, Berlin, DE). Magnetic glass particles (e.g. MPG (CPG Inc., Lincoln Park, New Jersey, US)) and magnetite-containing pigments (e.g. Microna Matte, Mica Black, Colorona Blackstar (all Merck)) can also be used as magnetic carriers. Non-porous magnetic particles (such as MagPrep) are particularly well suited because they cannot block pores, which would drastically impair the enzyme activity.

The enzyme carrier usually has the following properties: The particle size of the carrier is preferably 0.005 to 1 mm, more preferably 0.01 to 0.5 mm. The pore diameter is usually in the range from 10 to 4000 nm, a pore diameter from 30 to 100 nm being particularly preferred. A sufficiently large pore size can ensure that the enzyme fits on the support without loss of activity. The particle surface is advantageously 40 to 100 m ² / g, and the pore volume is preferably selected from a range of 0.5 to 3 ml / g. In some cases, a very large pore diameter of 2 to 20 μm can also be suitable.

The enzyme can be coupled covalently or adsorptively. Generally, a covalent coupling is preferable. Examples of a covalent coupling include epoxidation, a carbodiimide method, silanization, a cyanogen bromide method, a glutaraldehyde crosslinking or a dicresyl chloride method (see Biotransformations and Enzyme Reactions, Bommarius, AS, Biotechnology (2nd ed.), Vol. 3, p. 427-465, compiled by Stephanopoulos, G., VCH Weinheim, Germany 1993, Walt, DR et al., Trends in analytical chemistry, Vol. 13, No. 10, 1994, NH Park, HN Chang; J. Ferment. Technol ., Vol. 57 (4), 310-316, 1979, M. Puri et al .; Enz. Microb. Technol., 18, 281-285, 1996 and HY. Tsen; J. Ferment. Technol., 62 ( 3), 263-267, 1984). To carry out these processes, it is necessary for the support to be surface-modified with appropriate functional groups. The functional group can be carried out on the support either by copolymerization with functional monomers or by polymer analog conversion. A surface modification with amino residues, aldehyde groups, epoxy groups or a diol modification is particularly preferred. The enzymes can then be covalently bound to these groups.

The enzymatic hydrolysis takes place in a suitable reactor. A commercially available column is particularly suitable for a continuous implementation of the method according to the invention. On a small scale, for example, a column such as that used for preparative HPLC can be used. The reactor, especially the column, should have a high hydraulic efficiency. This can be quantified by the number of theoretical floors. It is therefore advantageous that the raw material solution comes into intensive contact with the surface of the property in order to ensure effective use of the enzyme and to achieve high productivity. The preparative HPLC column mentioned fulfills these requirements. and is also equipped with the appropriate technology with peripherals (pumps, valves, control). It is also advantageous that detection techniques such as UV or RI detection techniques have already been developed for this, so that, if desired, the measurement and regulation of the degree of conversion of the reaction can be carried out automatically.

If magnetic carrier materials are used in the continuous mode of operation, tubular reactors are usually used with a device which keeps the magnetic particles in suspension, e.g. electromagnetic coils that generate a largely homogeneous magnetic field in the flow tube, the field lines of which run parallel to the direction of flow (Helmholtz magnetic field). In such magnetically stabilized fluidized bed or fluidized bed reactors (Magnetically Stabilized Fluidized Bed (MSFB)), significantly higher flow rates can be achieved than in conventional fluidized beds or fluidized bed columns, but these are also suitable for these purposes. This technology can also be used advantageously for catalytic reactions in viscous reaction media.

A conventional container, which is preferably equipped with a stirring device, is suitable for carrying out the process discontinuously. A round bottom flask with a stirrer can be used on a small scale and a stirred kettle on a large scale.

The immobilizate is packed into the reactor in the usual way before the reaction.

The rutinoside to be reacted is usually fed into the reactor, for example a column, such as a fixed bed column, in the form of a solution or suspension. If the reactor is a fixed bed reactor, the rutinoside solution should be completely free of solids. It is advantageous to pre-dissolve the rutinoside with the solvent in a tank, preferably with stirring and / or heating, in order to achieve optimal solubility. If necessary, pre-filtration of the solution can also be performed to remove any solids. The solvent is preferably an aqueous system to ensure enzyme activity and to prevent possible denaturation. To ensure the solution of the rutinosides, NEN additional solvents are added. The process according to the invention is preferably carried out in the presence of a solvent mixture of water and at least one organic solvent.

The organic solvent or solvents additionally present include both organic solvents which are miscible with water and organic solvents which are not miscible with water.

Suitable solvents for the process according to the invention are nitriles, such as acetonitrile, amides, such as dimethylformamide, esters, such as acetic acid esters, in particular methyl acetate or ethyl acetate, alcohols, such as methanol or ethanol, ethers, such as tetrahydrofuran or methyl tert-butyl ether and hydrocarbons, such as Toluene. The process according to the invention is preferably carried out in the presence of one or more of the organic solvents acetic acid ester, methanol, ethanol, methyl tert-butyl ether or toluene. The process according to the invention is particularly preferably carried out in the presence of one or more acetic acid esters, in particular in the presence of methyl acetate, in addition to water.

Suitable volume ratios of water to organic solvent for the process according to the invention are ratios from 1:99 to 99: 1. The process according to the invention is preferably carried out with volume ratios of water to organic solvent from 20:80 to 80:20, in particular with volume ratios from 50:50 to 70:30.

The amount of rutinoside in the solvent or solvent mixture for the process according to the invention depends on the solubility of the rutinoside in the solvent or solvent mixture. In order to carry out the process according to the invention optimally, the rutinoside should be readily soluble. It is therefore preferable to work with an undersaturated solution. The amount of rutinoside in the solvent or solvent mixture is usually 0.001 to 5 g / l, preferably 0.05 to 2 g / l, more preferably 0.1 to 1.5 g / l. The ratio of rutinoside to immobilizate or enzyme depends on the life of the enzyme in the column and its activity in immobilized form.

The reaction is usually carried out at a temperature of 15 to 80 ° C. A temperature of 30 to 60 ° C. is preferred, in particular a temperature of 40 to 50 ° C. is advantageous in order to counteract possible destruction of the enzyme and at the same time to ensure a high solubility of the rutinoside

If the reaction temperature is too low, the reaction proceeds at an inappropriately slow reaction rate due to decreasing enzyme activity. In addition, the solubility of the rutinoside is reduced in such a way that unnecessarily high amounts of solvent are necessary. On the other hand, if the reaction temperature is too high, the enzyme, which is a protein, is denatured and thus deactivated.

If the process according to the invention is to be carried out at an elevated temperature, the reactor can be provided with a temperature control device. Common temperature control devices include a heating coil system or double jacket. It is furthermore advantageous if the rutinoside to be reacted and in particular the rutinoside solution is tempered before entering the reactor. For this purpose, it is common for the rutinoside solution to be fed from a temperature-controlled tank which is set to the temperature desired for the reaction. Alternatively, the solution to be fed can be passed through a heatable hose in order to set the desired temperature before entering the reactor. Possible tempering out of the rutinoside can also be avoided by the temperature control.

Suitable pH values for the method according to the invention are pH values between 3 and 8. The method according to the invention is preferably carried out at pH values from 3 to 7, in particular at pH values from 3 to 6. However, further preferred pH values can be used vary within the given limits depending on the enzyme used. For example, pH values from 3.8 to 4.3 are extremely preferred when using the enzyme hesperidinase. The method is preferably carried out in such a way that the pH is adjusted with the aid of a buffer system. In principle, all common buffer systems that are suitable for setting the above-mentioned pH values can be used. However, aqueous citrate buffer is preferably used.

The rutinoside mixture, which may be in the form of a solution or a suspension, is added to the reactor containing the immobilizate to carry out the enzymatic hydrolysis. This reaction can be batch, i.e. be carried out batchwise or continuously.

If the reaction is carried out batchwise, a rutinoside suspension is usually added to the reactor. The degree of conversion is determined by the amount of rutinoside and immobilizate. The ratio of rutinoside to immobilizate is usually 100: 1 to 1: 1000, preferably 10: 1 to 1: 100, more preferably 1: 1 to 1:20. The ratio of the immobilizate to the total volume of the suspension is usually 1: 1000 to 1: 1, preferably 1: 100 to 1: 2, more preferably 1:50 to 1: 5. The residence time in the reactor is normally 1 hour to 10 days, preferably 8 hours to 4 days, more preferably 1 to 2 days.

If the reaction is carried out continuously, a rutinoside solution is usually continuously and continuously conveyed through the reactor, preferably a column or MSFB reactor, using a suitable pump. By setting the flow rate accordingly, any desired degree of conversion can be achieved. Normally, a flow rate of 0.001 to 1 mm / s, based on the empty pipe cross-section of the column, is used.

Experience has shown that the activity of the enzyme in the system decreases over time. It is therefore necessary for the immobilisate to be replaced in whole or in part at regular intervals. In order to compensate for a loss of activity of the enzymes, it is advantageous to evaluate the degree of conversion by UV or RI detection, so that when the composition is changed, control via the pump output counteracts the change. After the reaction solution has emerged, the product obtained can be separated. After the reaction has ended, the reaction mixture consists mainly of solvent, unreacted rutinoside, rhamnose, the desired monoglycosidated flavonoid and possibly other additives, such as buffers. The monoglycosidated flavonoid usually precipitates as soon as the solubility limit is reached and gradually accumulates as a solid.

In the case of a discontinuous procedure with magnetic carrier materials, the immobilized enzyme can be separated from the product suspension in a simple manner with the aid of a magnetic separation device after the reaction has ended. A strong permanent magnet in plate form can be used on a laboratory scale. However, there are also larger separators that were developed for a wide variety of industrial applications and mostly work according to the HGMS principle (high gradient magnetic separation). Such a system can e.g. consist of a vertical flow tube, which contains a package of fine stainless steel wires. By means of suitably arranged electromagnetic coils, high magnetic field gradients are generated on the wires and a very effective separation of even the smallest particles in the nanometer range is achieved. If the magnetic particles are superparamagnetic, i.e. In the absence of an external magnetic field, there is no remanence magnetization, after switching off the magnetic field, it can be completely removed again from the separator by repeated rinsing with water.

The desired reaction product is isolated by customary methods with customary workup.

The product is preferably precipitated when concentrated. If the solvent contains a mixed solvent containing at least one organic solvent, it is preferred that the organic solvent be distilled off under reduced pressure. The crystallizing monoglycosidated flavonoid is usually separated from the remaining reaction mixture by solid / liquid separation, such as suction or filtration under reduced pressure or by centrifuging off the precipitated Crystals, separated. The solid is then washed, preferably with water and then dried.

Alternatively, the entire reactor contents can first be filtered off. The filter cake containing the product is then treated with a solvent or a buffer-solvent mixture in which the product is soluble. The reaction product is extracted from the filter cake.

In the case of a batchwise procedure, the catalyst, the immobilisate, which is insoluble in this mixture, remains. For this it is necessary that the solvent or the buffer-solvent mixture do not have an adverse effect on the enzyme. It has been shown that the immobilized enzyme, e.g. Naringinase or hesperidinase, in certain solvent-buffer mixtures or under moderately alkaline conditions, has no or only a fraction of the original activity, but the activity can be practically completely restored if the enzyme is subsequently carefully mixed with a buffer in the pH range 4 -6 is rinsed; it is therefore a temporary loss of activity, not an irreversible denaturation of the enzyme.

Extraction agents which are very suitable for this procedure are tetrahydrofuran buffer mixtures - preferably with a tetrahydrofuran content of 10-25%, in particular also at a slightly elevated temperature. Other suitable extractant components are e.g. 1-propanol, 2-propanol, 1, 4-dioxane and methyl acetate. The product can be very easily recovered from the extract by distilling off the solvent under reduced pressure and then cooling the aqueous product solution to 0 to 10 ° C. The reaction product crystallizes out of the mother liquor in very high purity.

As an alternative to solvent / buffer mixtures, a dilute ammonia or soda solution can also be used as the extraction agent, since the reaction product has phenolic OH groups which are already deprotonated in a weakly basic medium; the anion of the reaction product is comparatively readily soluble, but is also very sensitive to oxidation, which is the result of a gradual discoloration of the extract from yellow to brown. Therefore, this variant should be operated quickly, ie the extraction should be completed within 10 minutes to 6 hours, preferably 20 minutes to 2 hours. It is preferable to work in a protective gas atmosphere.

The enzyme activity is not lost even by treatment with weakly basic extraction agents, such as aqueous solutions of alkali or ammonium salts of acetic acid, oxalic acid, citric acid, phosphoric acid, boric acid or carbonic acid, or aqueous solutions of alkylamines, piperidine or pyridine. The reaction product can be precipitated again by acidifying the extract and cooling to 0-10 ° C.

The purity of the monoglycosidated flavonoid obtained using pure rutinoside is normally greater than 94%. For further purification, the end product can, for example, be recrystallized from suitable solvents, e.g. from water or from solvent mixtures consisting of toluene and methanol or water and methyl acetate.

The amount of solvent obtained after the reaction is preferably recovered in order to ensure the economy of the process according to the invention. This recirculation is usually continuous and automated. Commercial evaporator systems with appropriate controls are available for this. If the solvent to be used is a solvent mixture of water and at least one organic solvent, it is generally not possible to immediately use the distillate again for the process, since the solvent ratios have changed as a result of the organic solvent being distilled off. The desired solvent ratio can be restored by automatic quality measurement and correction and the solvent can thus be returned.

Furthermore, membrane processes or nanofiltration can be carried out during the concentration. In this process, the solvent mixture is separated without changing the composition. The following examples are intended to illustrate the present invention. However, they are in no way to be regarded as restrictive.

example 1

Immobilization of the enzyme hesperidinase on a silica gel support

1) Carrier surface conditioning for immobilization

1.1) Properties of the carrier material silica gel LiChrospher diameter = 15 - 40 μm pore diameter = 300 A particle surface = 80 m ² / g pore volume = 0.73 ml / g density = 2 g / ml

1.2) Silica activation

250 g of silica gel are mixed with sufficient HCI (7%) in a 1 L bottle and left overnight to moisten the silica gel.

The silica gel suspension is then washed chloride-free with demineralized water. To do this, you have to test the supernatant with nitric acid and silver nitrate after each wash. Because of the properties of the silica gel particles, the washing is carried out on an approximately 24 cm diameter ceramic funnel.

1.3) Surface modification with amino groups

The acid-treated silica gel is mixed with enough water in a 2 L three-necked flask equipped with a reflux condenser and a dropping funnel, so that it becomes stirrable. At room temperature and good mixing 1 mmol / g carrier of the 3-aminopropyltrimethoxysilane (for 250 g of silica gel, 135 ml of solution is required) is added dropwise to the silica gel suspension at a rate of about 5 drops / s. The suspension is then stirred at 90 ° C. for 2 hours. The suspension is then cooled with ice.

The supernatant must be checked for possible residues of 3-aminopropyltrimethoxysilane by means of pH measurement. The bead suspension is washed with demineralized water until the pH remains constant.

14) Glutardialdehyde coverage

Glutardialdehyde (GDA) is added to the silica gel suspension obtained in a concentration of 1 mmol / g of carrier (13 ml of 50% GDA solution are required for 250 g of carrier). The suspension (plus some water) is rolled in a 1 L bottle at room temperature for 2 hours. At the beginning the suspension is colored yellow and at the end it turns dark red.

The supernatant from each wash is then checked for residues of glutardialdehyde by a precipitation reaction with dinitrophenylhydrazine. The suspension is washed carefully until the test is negative.

2) Immobilization

2.1) hesperidinase

First addition of protein

3.8 g of hesperidinase are stirred in 500 ml of citrate / phosphate buffer (pH = 6.0). 300 μl of surfactants (Tween 20) are added for better dissolution. The enzyme solution is then filtered. In a 1 L bottle, approx. 230 g of the silica gel suspension obtained under 1.4) are mixed with the enzyme solution. The enzyme carrier suspension is then rolled at room temperature for about 40 hours.

Second protein addition

Approx. 0.76 g hesperidinase (Amano) are stirred in 120 ml citrate / phosphate buffer (pH = 6.0) with 60 μl surfactants and later filtered.

The enzyme solution is poured into the 1 L bottle mentioned above, and the enzyme solution is rolled at room temperature.

2.2) BSA (for the separation test)

0.3 Biomex BSA (bovine serum albumin powder) is stirred in 100 ml citrate / phosphate buffer (pH = 6.0). Approx. 20 g of the silica gel suspension obtained under 1.4) are mixed with the protein solution in a 0.5 L bottle, and 60 μl ProClin300 are added.

3) Protein quantity and activity determination

3.1) Amount of protein (mg protein / ml)

The protein content of a solution is determined using the Bradford test. The standard assay is performed. 20 μl sample are mixed in 1 ml Bradford dye reagent (diluted 1: 5) and measured after 15 min in a photometer at 595 nm.

The microassay must be carried out at very low protein concentrations. 0.8 ml of sample are mixed in 0.2 ml of Bradford dye reagent (concentrated) and measured after 15 min in a photometer at 595 nm. 3.2) Activity

The activity of a solution is measured by the reaction with a replacement substrate. For each sample one takes:

88 ul citrate / phosphate buffer (pH = 4.0) 100 ul sample 20 ul sample

20 μl replacement substrate: p-nitrophenyl-α-L-rhamnoside (rhamnosidase activity) p-nitrophenyl- -L-glucoside (glucosidase activity)

This 1 ml solution is mixed in an Eppendorf reaction vessel. After an incubation time of 2 and 5 min at 40 ° C in a shaker, 100 μl of the reaction mixture are mixed with 1 ml of 1 M soda solution. Then the concentration of p-nitrophenol is measured in the photometer at 400 nm. The activity is calculated by changing the concentration of p-nitrophenol per time.

The activity of an enzyme is given in units (U) (= μmol converted substrate per minute).

4) Results

PROTEIN CONTENT and ACTIVITY VALUES (FREE HESPERIDINASE)

¹ HespO: first protein addition Hespl: second protein addition Samples 1-6: supernatant

PROTEIN CONTENT and ACTIVITY VALUES (IMMOBILIZED HESPERIDINASE)

Protein content of the carrier ≤ 1, 045 g bound protein

There are 4.7 mg protein / g carrier on the carrier

2% of the added protein was not bound Example 2

Preparation of isoquercetin from rutin by enzymatic hydrolysis using an immobilizate.

3200 l of demineralized water and 800 l of 1-propanol are placed in a heatable 4.5 m ³ stirred tank (1). The mixture is heated to approx. 50-60 ° C via the steam supply (2). 8000 g of rutin, DAB are added to the solution with vigorous stirring. The mixture is stirred until the rutin has completely dissolved. The pH value is then checked using a circulating pump and a pH meter (3a) installed in the line and adjusted to pH 4.0 - 4.5 if necessary (with H ₃ P0 ₄ and NaOH). A sample can also be taken via the manual valve (4) for checking and determining the concentration.

At the start of the reaction, the solution is fed to the piston metering pump (7) via a bag filter (5) and a candle filter (6). The bag filter has the task of stopping the larger amount of undissolved components, while the candle filter cleans the solution with a fineness of 0.2 μm.

The piston metering pump (7) conveys the solution through a heatable hose, which adjusts the solution to a temperature of 40 ° C at the entrance of the column using a thermometer, with a flow of 1 l / min into the column (9) (100x400 mmj. The column contains 1.5 kg of immobilized material, since the electrically heated hose cannot cool the solution, the temperature in the stirred tank (1) must be selected so that cooling on the way to the pump leads to a maximum pump flow of up to 40 ° C.

A sample can be taken from the solution after passing through the column via a manual valve (10), with which the temperature and the degree of conversion of the reaction can be measured off-line again. If the measured degree of conversion is lower than required, the flow rate of the pump is reduced accordingly.

After passing through the column, the reaction is complete, so that the solution can be fed into the collecting vessel (11). There the solution is about 10-20% of the volume is removed from the capacitor (12). This significantly reduces the propanol content, which greatly reduces the solubility of the isoquercetin. Subsequent cooling further reduces the solubility, so that the product precipitates and can be separated in the prey filter (13). From there it is transferred to a drying cabinet (14) for further drying. The mother liquor and the distilled-off condensate are returned together in the stirred tank (1) for fresh preparation.

Example 3

1 Modification of silica gel particles with aldehyde groups and immobilization of naringinase on these particles

250 g of silica gel (eg LiChrospher Sl 300, Merck, Darmstadt) were poured with 400 ml of 10% HCl in a closable vessel, degassed with ultrasound for 10 minutes and left to stand at RT for 24 hours. The silica gel was then filtered off and demineralized with several liters. Washed water until the pH was> 4.5 and chloride ions were no longer detectable in the filtrate (spot reaction with acetic acid AgN0 ₃ solution).

The acid-treated moist silica gel was then transferred to a 4 1 three-necked flask equipped with KPG stirrer, reflux condenser and 100 ml dropping funnel and slurried with 3 l of deionized water. With stirring, 100 ml of aminopropyltrimethoxysilane (ABCR, Karlsruhe) were added via the dropping funnel over the course of 15 minutes. The suspension was then heated and stirred at 90 ° C. for 90 minutes. The cooled suspension was filtered and eight times with 1 l of demin. Washed water.

The amino-activated silica gel was suspended in 3 I using ultrasonically degassed water in a 4 I three-necked flask equipped with KPG stirrer and 100 ml dropping funnel; the pH was lowered to 8.0 with a few drops of 2 M acetic acid. Then 100 ml of 50% glutardialdehyde solution (Merck, Darmstadt) were added dropwise within 1 h and the suspension was stirred for a further 2.5 h at RT. The activated silica gel is filtered again and so long with ice-cold demin. Washed water until Wash water no more glutaraldehyde could be detected (spot reaction with sulfuric acid 2,4-dinitrophenylhydrazine solution).

The silica gel modified with aldehyde groups was deminized in 500 ml. Water is suspended in a 4 liter flask by stirring with a KPG stirrer. 13 g of naringinase (Sigma, Deisenhofen) were dissolved in 2.5 1 0.25 M phosphate buffer, pH 8.0. The enzyme solution was added to the silica gel suspension and stirred at RT for 96 h. The immobilizate was then filtered off and washed several times, first with 0.2 M sodium chloride solution and then with 50 ml of citrate buffer, pH 4.0. The rhamnosidase activity of the immobilizate was determined using p-nitrophenyl-L-α-rhamnopyranoside (Sigma, Deisenhofen) as substrate by the Kurosawa method; it was 120 U / g.

2. Immobilization of hesperidinase on Eupergit ™ C

50 g of Eupergit (Röhm, Weiterstadt) were mixed in a screw-on 500 ml glass bottle with 300 ml of 0.8 M potassium phosphate buffer, pH 8.5, and left to stand for 30 minutes. Then 5.0 g of hesperidinase (Amano) were added and the mixture was stirred for 120 hours at RT on a roller mixer. The Eupergit was filtered off by means of a glass frit and washed several times first with 0.2 M sodium chloride solution, then twice with 1 I 0.1 M citrate buffer, pH 4.0. The rhamnosidase activity of the immobilizate was determined using p-nitrophenyl-L- rhamnopyranoside (Sigma, Deisenhofen) as substrate by the Kurosawa method; it was 15 U / g, based on dry immobilizate, or 4.2 U / g, based on moist immobilisate.

3. Conversion of rutin to isoguercetin with hesperidinase immobilized on Eupergit in a stirred tank reactor and subsequent extraction of the product with a tetrahydrofuran / buffer mixture

1000 ml of 50 mM citrate buffer, pH 4.0, 100 g (wet weight) immobilized on Eupergit with an activity of 4.2 U / g, and 10 g of rutin (Merck, Darmstadt) at 40 ° C. were placed in a 2000 ml round-bottomed flask stirred with a KPG stirrer; the degree of turnover was continuously determined by HPLC analysis. After a total of 96 h the reactor contents were filtered off via a Büchner funnel. The filter cake was returned to the round bottom flask and stirred for 30 minutes in a mixture of 400 ml of 50 mM citrate buffer, pH 4.0 and 100 ml of tetrahydrofuran at 40 ° C., with most of the isoquercetin dissolving. The mixture was filtered warm and the filter cake extracted again with 500 ml of buffer tetrahydrofuran mixture for 30 minutes. After filtration, the two isoquercetin extracts were combined with the first filtrate and the tetrahydrofuran was removed using a rotary evaporator. In order to completely precipitate the product, the aqueous isoquercetin solution was cooled to 4 ° C. After filtration and drying in a desiccator, a yield of 5.8 g of product was obtained, which was composed of 98% isoquercetin and 2% rutin.

The moist eupergite was washed once with cold tetrahydrofuran buffer mixture, then repeatedly with 50 mM citrate buffer, pH 4.0, until the smell of tetrahydrofuran was barely perceptible. The activity of the enzyme was still 3.6 U / g, which corresponds to an activity loss of 14%.

4. Conversion of rutin to isoguercetin with hesperidinase immobilized on Eupergit in a stirred tank reactor and subsequent extraction of the product with an alkaline buffer solution

1000 ml of 50 mM citrate buffer, pH 4.0, 100 g of naringinase immobilized on Eupergit with an activity of 4.2 U / g (wet weight) and 10 g of rutin (Merck, Darmstadt) at 40 ° C. were placed in a 2000 ml round-bottomed flask stirred with a KPG stirrer; the degree of turnover was continuously determined by HPLC analysis. After a total of 96 h the contents of the reactor were filtered off via a Buchner funnel. The moist filter cake was put back into the round bottom flask and stirred at RT in 300 ml of 50 mM sodium carbonate buffer, pH 10.0 for 5 min, during which part of the isoquercetin dissolved with an intensely yellow color. The suspension was filtered and the filter cake was immediately extracted again with carbonate buffer. After a total of 7 extraction cycles, the eupergite was largely colorless and the isoquercetin was almost completely dissolved. The extracts were combined, carefully acidified with dilute hydrochloric acid until the pH was approximately 3 and then cooled to 4 ° C. After filtration and drying in a desiccant A yield of 4.9 g of product was obtained, which was composed of 98% isoquercetin and 2% rutin.

The moist eupergite was washed twice with 50 mM citrate buffer and was then ready for further reactions. The activity of the enzyme was still 3.9 U / g, which corresponds to a loss of activity of 7%.

Example 4

1 Modification of magnetic silica particles with aldehyde groups and immobilization of naringinase on these particles

A suspension of 30 g magnetic silica particles (MagPrep, Merck, Darmstadt) in 600 ml water was placed in a 1 liter three-necked flask equipped with a KPG stirrer, dropping funnel and reflux condenser. A mixture of 20 ml of aminopropyltriethoxysilane (ABCR, Karlsruhe) and 20 ml of isopropanol was added dropwise with stirring over the course of 30 minutes. The mixture was then heated to 85 ° C. and stirred at this temperature for 1 hour. After cooling, the suspension was transferred to a beaker, the particles were collected at the bottom of the vessel using a strong permanent magnet, and the supernatant was decanted. The particles were repeated with demin. Washed water until the pH of the wash water remained constant. The particles were then resuspended in 600 ml of water and the pH was adjusted to about 8 with a few drops of acetic acid; after the addition of 24 ml of 50% glutardialdehyde solution, the suspension was stirred at RT for 4 h and the particles thereafter with demin. Washed water until no more glutaraldehyde could be detected in the wash water (spot reaction with sulfuric acid 2,4-dinitrophenylhydrazine solution).

The aldehyde-derivatized particles were resuspended in a 1 liter round-bottom flask in 600 ml of 0.2 M potassium phosphate buffer, pH 9. After adding a solution of 1 g of naringinase (Sigma, Deisenhofen) in 100 ml of 50 mM sodium chloride solution, the mixture was stirred at RT for 2 days using a KPG stirrer. The particles were then separated using a permanent magnet and repeated first with 0.2 M sodium hydroxide. rium chloride solution, then washed with 50 mM citrate buffer, pH 4.0. The rhamnosidase activity of the immobilizate was determined using p-nitrophenyl-L- rhamnopyranoside (Sigma, Deisenhofen) as a substrate using the Kurosawa method (Kurosawa, Ikeda, Egami, J. Biochem. 73, 31-37 (1973) : α-L-Rhamnosidases of the liver of Turbo cornutus and Aspergillus niger); it was 162 U / g.

2. Modification of magnetic silica particles with epoxy groups and immobilization of naringinase on these particles

A suspension of 30 g of magnetic silica particles (MagPrep, Merck, Darmstadt) in 600 ml of 50 mM sodium acetate solution was placed in a 1 liter three-necked flask equipped with a KPG stirrer, dropping funnel and reflux condenser. A mixture of 20 ml (3-glycidoxypropyl) trimethoxysilane (ABCR, Karlsruhe) and 20 ml of isopropanol was added dropwise over the course of 30 minutes with stirring, then the mixture was heated to 85 ° C. and stirred at this temperature for 1 hour. After cooling, the suspension was transferred to a beaker, the particles were collected at the bottom of the vessel using a strong permanent magnet, and the supernatant was decanted. The particles were repeated with demin. Washed water until the pH of the wash water remained constant. For quantitative determination of the epoxy groups, a sample of about 0.5 g of the material was washed repeatedly with methanol and then dried to constant weight at about 70 ° C. in a drying cabinet. The determination of the epoxy groups by the method of Pribyl (Pribyl, Fresenius Z. Anal. Chem. 303, 113-116 (1980): determination of epoxy end groups in modified chromatographic sorbents and gels) gave 250 μmol / g.

150 ml of a 20% (w / v) suspension of epoxy-derivatized magnetic particles were mixed in a 1 l round-bottom flask with 350 ml of 1 M potassium phosphate buffer, pH 9.0. After adding a solution of 1.5 g of naringinase (Sigma, Deisenhofen) in 15 ml of 50 mM sodium chloride solution, the mixture was stirred at 40 ° C. for 16 h using a KPG stirrer. The particles were then separated using a permanent magnet and repeatedly washed first with 0.2 M sodium chloride solution, then with 50 mM citrate buffer, pH 4.0. The rhamnosidase activity of the immobilizate was determined using p-nitrophenyl-L- α-rhamnopyranoside (Sigma, Deisenhofen) determined as substrate by the Kurosawa method; it was 102 U / g.

3. Modification of magnetic silica particles with carboxyl groups and immobilization of naringinase on these particles

A suspension of 30 g magnetic silica particles (MagPrep, Merck, Darmstadt) in 600 ml water was placed in a 1 liter three-necked flask equipped with a KPG stirrer, dropping funnel and reflux condenser. A mixture of 28 ml of 3- (triethoxysilyl) propylsuccinyl anhydride (ABCR, Karlsruhe) and 28 ml of isopropanol was added dropwise with stirring over the course of 30 minutes and then the pH of the reaction mixture was adjusted to 9.0 by dropwise addition of a 10% sodium hydroxide solution , The mixture was heated to 80 ° C and stirred at this temperature for 2 hours. The pH was checked at regular intervals and corrected if necessary by adding alkali. After cooling, the suspension was transferred to a beaker, the particles were collected at the bottom of the vessel using a strong permanent magnet, and the supernatant was decanted. The particles were demineralized three times. Water, once with a 2 M acetic acid solution and then repeatedly with demin. Washed water until the pH of the wash water remained constant.

150 ml of a 20% (w / v) suspension of carboxyl-derivatized magnetic particles were in a 1 l round-bottom flask with 300 ml of 0.4 M potassium phosphate buffer, pH 5.0, and a solution of 1.5 g of naringinase (Sigma, Deisenhofen) mixed in 150 ml of 50 mM sodium chloride solution. After adding 8 ml of a 1% (w / v) solution of EDC (N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride, Merck, Darmstadt) in water, the mixture was stirred at RT for 20 h. The particles were then separated using a permanent magnet and repeatedly washed first with 0.2 M sodium chloride solution, then with 50 mM citrate buffer, pH 4.0. The rhamnosidase activity of the immobilizate was determined using p-nitrophenyl-L-α-rhamnopyranoside (Sigma, Deisenhofen) as substrate by the Kurosawa method; it was 71 U / g. 4. Modification of magnetic mica pigments with aldehyde groups and immobilization of hesperidinase on these particles

A suspension of 30 g of magnetic mica pigments ("Colorado Blackstar Green", Merck, Darmstadt) in 300 ml of water was placed in a 1 liter three-necked flask equipped with a KPG stirrer, dropping funnel and reflux condenser. A mixture of 20 ml of aminopropyltriethoxysilane (ABCR, Karlsruhe) and 20 ml of isopropanol was added dropwise with stirring over the course of 30 minutes. The mixture was then heated to 85 ° C. and stirred at this temperature for 1 hour. After cooling, the suspension was transferred to a beaker, the particles were collected at the bottom of the vessel using a permanent magnet, and the supernatant was decanted. The particles repeated with demin. Washed water until the pH of the wash water remained constant. The particles were then resuspended in 300 ml of water and the pH was adjusted to about 8 with a few drops of acetic acid; after addition of 25 ml of 50% glutardialdehyde solution, the suspension was stirred for 4 hours at RT and the particles thereafter with demin. Washed water until no more glutaraldehyde could be detected in the wash water (spot reaction with sulfuric acid 2,4-dinitrophenylhydrazine solution).

30 g of aldehyde-derivatized mica pigments "Colorona Blackstar Green" were resuspended in a 1 liter round-bottom flask in 300 ml of 0.2 M potassium phosphate buffer, pH 7.5. After adding a solution of 1 g hesperidinase (Amano) in 20 ml 0.2 M potassium phosphate buffer, pH 7.5, the mixture was stirred with a KPG stirrer for 3 days at RT. The particles were then separated using a permanent magnet and repeatedly washed first with 0.2 M sodium chloride solution, then with 50 mM citrate buffer, pH 4.0. The rhamnosidase activity of the immobilizate was determined using p-nitrophenyl-L-α-rhamnopyranoside (Sigma, Deisenhofen) as substrate by the Kurosawa method; it was 10 U / g. 5. Conversion of rutin to isoguercetin with immobilized naringinase in the stirred tank reactor and separation of the magnetic biocatalyst with a permanent magnet

400 ml of 50 mM citrate buffer, pH 5.0, 20 g of naringinase immobilized on magnetic silica particles with an activity of 102 U / g and 10 g of rutin (Merck, Darmstadt) were stirred together at 40 ° C. in a 500 ml double-jacket stirred reactor; the degree of conversion was determined at intervals by HPLC analysis. After 24 hours, the reactor contents were pumped into a beaker and the catalyst was collected at the bottom of the vessel using a plate magnet (Bakker, 200 mT). The supernatant was immediately aspirated using a pump and the magnetic particles were washed several times with 100 ml of buffer in order to rinse off the last residues of adhering solid isoquercetin. The collected isoquercetin was filtered off, washed several times with small portions of ice water and dried in a desiccator. The yield was 6.5 g. HPLC analysis showed a composition of 96% isoquercetin, 2% quercetin and 2% rutin. The activity of the immobilized product was still 92 U / g after the reaction. This corresponds to an activity loss of 10%.

6. Conversion of rutin to isoquercetin with immobilized naringinase in a stirred tank reactor and separation of the magnetic biocatalyst with an electromagnetic separator (Fig. 1)

300 ml of 50 mM citrate buffer, pH 5.0, 10 g of naringinase immobilized on magnetic silica particles with an activity of 102 U / g and 5 g of rutin (Merck, Darmstadt) were stirred together at 40 ° C. in a 500 ml double-jacket stirred reactor. the degree of turnover was continuously determined by HPLC analysis. After 24 hours, the reactor contents were passed through an electromagnetic HGMS system using a peristaltic pump with a flow rate of 25 ml / min, whereby the magnetic particles were completely separated on the wire matrix (technical data of the separation system: glass tube with an inner diameter of 20 mm and 200 mm Length, empty volume 65 ml, weight of the wire packing made of SS alloy 15 g, 4 coils connected in series, current 6 A, magnet. Field strength of the Helmholtz field 25 mT). Subsequently, 100 ml of citrate buffer were pumped through the column with the magnetic field switched on in order to rinse the magnetic particles. The combined product suspensions were filtered, the isoquercetin washed with ice water and dried in a desiccator. The yield was 3.1 g. HPLC analysis showed a composition of 97% isoquercetin, 2% quercetin and 1% rutin.

To recover the catalyst, 100 ml of citrate buffer, pH 5.0, were pumped through the separator at a flow rate of 100 ml / min for 10 minutes with the magnetic field switched off, the direction of flow being changed several times. The catalyst suspension was then pumped back into the stirred tank and the remaining amount of catalyst remaining in the separator was rinsed again twice with 100 ml of citrate buffer. The activity of the immobilizate was still 94 U / g after the reaction. This corresponds to an activity loss of 8%.

7. Conversion of rutin to isoquercetin with hesperidinase immobilized on mica particles in a stirred tank reactor and separation of the magnetic biocatalyst using a plate magnet

400 ml of 50 mM citrate buffer, pH 5.0, 30 g immobilized on "Colorona Blackstar" with an activity of 10 U / g and 5 g of rutin (Merck, Darmstadt) at 40 ° C. were immobilized in a 500 ml double-jacket stirred reactor stirred; the degree of turnover was continuously determined by HPLC analysis. After 24 hours, the reactor contents were pumped into a beaker and the catalyst was collected at the bottom of the vessel using a plate magnet (Bakker, 200 mT). The supernatant was immediately aspirated using a pump and the magnetic particles were washed several times with 100 ml of buffer in order to rinse off the last residues of adhering solid isoquercetin. The collected isoquercetin was filtered off, washed several times with small portions of ice water and dried in a desiccator. The yield was 3.3 g. HPLC analysis showed a composition of 96% isoquercetin and 4% rutin. The activity of the immobilizate was 9.7 U / g after the reaction. This corresponds to a loss of activity of 3%.

8. Conversion of rutin to isoguercetin with naringinase immobilized on magnetic silica gel particles in an MSFB reactor

A mixture of 5 g of rutin, 900 ml of 50 mM citrate buffer, pH 5.0 and 100 ml of methyl acetate was stirred in a storage vessel at 40 ° C. until an approximately homogeneous suspension without larger agglomerates was formed. The reactor could not be heated. The methyl acetate was added to increase the solubility and redissolving rate at RT and to prevent the formation of quercetin. In the meantime, a suspension of 6 g of naringinase immobilized on magnetic silica particles with an activity of 162 U / g in 60 ml of 50 mM citrate buffer, pH 5.0, was conveyed into the tube of the MSFB reactor using a pump with the magnetic field switched off. A 20 mT magnetic field was then set and fresh citrate buffer was initially introduced from below with the aid of a precisely metered piston stroke pump with a flow of 5 ml / min until the particles had stabilized in the magnetic field. The rutin suspension was then pumped through the MSFB reactor at RT within 3.5 h. The methyl acetate was first removed from the product mixture on a rotary evaporator; then the product was filtered off, washed several times with ice water and dried in a desiccator. The product yield was 2.9 g; the product consisted of 86% isoquercetin and 14% rutin.

Claims

claims

1. A process for the preparation of monoglycosidated flavonoids by enzymatic hydrolysis of rutinosides, characterized in that an enzyme which is immobilized on a support is used for the enzymatic hydrolysis.

2. The method according to claim 1, characterized in that a rutinoside of the formula (A)

wherein RH, OH or OCH ₃ is used.

3. The method according to claim 1 or 2, characterized in that rutin is used as rutinoside.

4. The method according to any one of claims 1 to 3, characterized in that an α-L-rhamnosidase is used as the enzyme.

5. The method according to any one of claims 1 to 4, characterized in that hesperidinase is used as the enzyme.

6. The method according to any one of claims 1 to 5, characterized in that the enzyme is immobilized on silica gel.

7. The method according to any one of claims 1 to 5, characterized in that the enzymatic hydrolysis is carried out in the presence of a solvent mixture of water and at least one organic solvent.

8. The method according to any one of claims 1 to 7, characterized in that the reaction is carried out at a reaction temperature of 15 to 80 ° C.

9. The method according to any one of claims 1 to 8, characterized in that the reaction is carried out at a pH of 3 to 8.