CZ2018352A3 - Heterogeneous process for producing rutinose - Google Patents

Abstract

The object of the present invention is a heterogeneous process for the production of rutinose from rutin in a heterogeneous system, wherein rutin in the form of a suspension in aqueous medium at a pH ranging from 2.05 to 10.1 and at a temperature of 10 to 70 ° C is treated with rutinosidase enzyme. rutinose and quercetin, followed by separation of quercetin from the reaction mixture by centrifugation, filtration or decantation, and rutinose being obtained by crystallization or evaporation of the reaction mixture after separation of the solids.

Description

The invention relates to a heterogeneous enzymatic preparation of rutinose (α-L-rhamnopyranoside- (1-> 6) -D-glucopyranose) from the starting material rutin by glycosidase, obtained from microorganisms of the genus Aspergillus. An appreciated byproduct of the reaction is quercetin (2- (3,4-dihydroxyphenyl) -3,5,7trihydroxy-4H-chromen-4-one) of high purity, which is an effective antioxidant and is useful in nutraceuticals and other protective products (eg cosmetics, chemoprotective substances and functional foods).

BACKGROUND OF THE INVENTION

Flavonoids, which are abundant in plants, are generally considered to be effective antioxidants and chemoprotective agents. Typical components of natural flavonoids are their polyphenolic aglycones and glycosidic substituents. The most common glycones in natural flavonoids include D-glucose, L-rhamnose, D-galactose, D-xylose, L-arabinose, both in the form of monoglycosides, diglycosides and, rarely, more complex oligoglycosides. One of the most abundant polyphenolic aglycons is quercetin (see below).

Typical representatives of rutinosylated flavonoids are, for example, rutin and hesperidin, which are found in large quantities in citrus, buckwheat and many other plant sources. Rutin is produced in multi-ton quantities and is widely used in pharmaceuticals and dietary supplements. Both components of rutin - quercetin and rutinose - have extensive use and very interesting biological applications. Technologically simple, biocompatible and high-yield production of both of these components (ideally in a one-step reaction) would be a significant technological advance.

Rutinosa

Rutinose (and -L-rhamnopyranoside- (1-> 6) -D-glucopyranose, bound to polyphenol by a β-glycosidic bond) is a frequently occurring disaccharide in flavonoids, whereas quercetin (see below) is well studied and its use is extensive and well-documented, then the second component of rutin - rutinose - is still waiting for its use to a large extent.One of the main reasons for limited use of rutinosis is its poor availability - in catalogs of reputable chemical companies is offered in milligrams at excessive prices as an analytical standard. which does not allow practical application at all (25 mg at 10 000 CZK, Sigma-Aldrich).

The greatest potential for using rutinosa is in the cosmetics industry. Rhamnosa, located at the non-reducing end of rutinosis, is already widely used in cosmetics, such as LOreal, to eliminate or reduce wrinkle formation. The mechanism of action of rhamnose and rhamnooligosachaides composed of rhamnose, glucose, fucose and other carbohydrates is based on the principle of inhibition of elastase and other metalloproteinases in fibroblasts. The extent of the anti-aging effect depends on the structure of the selected rhamnooligosaccharides. It has been shown that L-rhamnose is bound by α-glycosidic bond to various sugar structures (as well as in rutinose), an essential structural element necessary for effect on fibroblasts, reduction of elastin degradation and other complex effects. It is further known that derivatization of rhamnose at the reducing end improves its bioavailability when topically applied via the epidermis to keratinocyte cells where it then acts. LRhamnosa is a carbohydrate common in the plant kingdom, but does not occur in vertebrates. However, it interacts with some lectin receptors in epidermis cells based on lectin interactions (causing the above-mentioned "anti-aging" effect), but is not actively transported to the cells. On the contrary, D-glucose is the basis of the energy metabolism of cells and the best energy source and practically

All cells import it very actively using a series of glucose transporters. This effect is also known for glucosides which are actively imported into the cells by the above-mentioned glucose transporters, and the whole glucoside then enters the cells at a much higher concentration, thus allowing a substantial increase in effect. Glucosides also exhibit generally lower toxicity and thus substantially reduce the possibility of adverse side effects.

Relatively complicated methods for the preparation of rutinosis by chemical synthesis are described in the literature. One of the first works on this topic practically only demonstrates the presence of rutinose in rutin by complicated sequential chemical degradation of rutin (J. H. Looker et al. Carbohydrate Reseach 13, 179, 1970). Furthermore, a series of (regio) isomers of rutinose were prepared by complex multi-step chemical synthesis with low overall yields, sufficient for the spectral characterization of the products in 1959 by P.A.J. Gorin and A.S. Perlin (Canadian Journal of Chemistry 37, 1930, 1959) and then later by another total synthesis of L. Birkofer et al. (Justus Liebigs Annalen der Chemie, 1973, 731, 1973). A small amount of rutinose besides methylrutinoside was prepared as novel substances by extraction from Datisca glomerata and subsequent multi-step isolation, including column chromatography and other procedures by M. Schubert et al. (Planta 231,507, 2010). In this work, it has been shown that rutinose has a similarly important role in the storage and transfer of energy substances as some of the more common sucrose in some plants.

Several attempts to prepare rutinose by enzymatic cleavage are described below, but these are complicated by the need to add toxic organic solvents, such as that described by Simčíková et al. (Advanced Synthesis Catalysis 357, 107-117, 2015) - here the rutin was dissolved in a mixture of dimethylsulfoxide and buffer, and after rutinosidase treatment, the cleaved rutinium had to be purified by silica gel column chromatography using organic solvents in a total yield of 63%.

In summary, no published method describes the direct preparation of pure rutinose without the need for, for example, column chromatography. Total synthesis methods are multi-step and provide rutinium in low yields, the described enzyme methods using either organic cosolvents or complex enzyme preparations from buckwheat, which, due to contamination with other monoglycosidases, provide an impure preparation, typically containing contaminating monosaccharides that needs to be further chromatographed. All the methods described work exclusively with a solution of rutin at very low concentrations (up to 5 g / L) and, moreover, do not solve the parallel recovery of quercetin at all. None of the described methods allow for the recycling of the enzyme due to inherent reasons.

Kvercetin

Flavonol quercetin is one of the most common and abundant flavonoids in the plant kingdom. Many flavonoids are present in natural sources in the form of glycosides. Quercetin and its glycosides quercetin-3-3-D-glucopyranoside (isoquercitrin) and quercetin-3-rutinoside (rutin) have a wide range of positive effects on the human body. The structure of quercetin is quite unique: the catechol moiety (ortho-z-dihydroxybenzene) together with the free hydroxy group on carbon C-3, which is conjugated to the 4-oxo group and delocalizes electron density from ring B and further configures the hydroxy groups to C-3 , -5 and -7 together with the 4-oxo group make quercetin a unique antioxidant. It is the free C-3 hydroxy group, which is very often glycosylated in natural derivatives of quercetin (eg rutin, quercitrin, isocvercitrin) is essential for the antioxidant activity of quercetin, therefore, the aglycone quercetin generally has higher antioxidant effects than its glycosides. Due to the fact that quercetin occurs almost exclusively in the form of the above mentioned and other glycosides in natural sources, it is necessary to release this aglycone from the respective glycosides in order to achieve the highest antioxidant, chemoprotective and other beneficial effects. Quercetin was previously considered potentially harmful due to the positive reaction in the Ames test, which implies mutagenicity and possible carcinogenicity of this substance (Manach et al., Nutr. Res. 16, 517, 1996). However, these doubts have recently been dispelled and the Federal Drug Agency (USA) has released a GRAS Notice No. 1 for quercetin. GRN 000341 (2010) - Generally Recognized

-2GB 2018 - 352 A3 as Safe. This certification then allowed a significant expansion of the commercial use of this substance in many different preparations, not only for special human nutrition, but also in veterinary preparations.

Rutin is commonly prepared by extraction from natural materials. This substance is a common pharmaceutical commodity and is widely used in many therapeutic applications, eg in the treatment of vascular capillary fragility, cerebral thrombosis, retinitis and as a systemic chemoprotectant. Quercetin is typically present in conventional crops of the western diet (eg onions, apples), mostly in the form of various glycosides, but it is difficult to obtain directly from natural sources and in poor purity, so these sources for the production of functional foods are virtually impossible use.

Extraction of quercetin itself from natural sources (eg onion - KR 2003016191-A) is economically completely incompetent, especially due to the low yields and the complexity of the procedures; another method described in Russian patents (RU 2182906-C1) is the oxidation of taxifoline (dihydrocvercetine), which is obtained by extraction from certain Siberian trees and subsequent oxidation with sodium nitrite. Even this multi-stage method does not provide a very pure preparation, it uses toxic chemicals and is not economical.

Quercetin is currently commonly prepared by chemical hydrolysis of rutin, typically boiling for several hours with dilute hydrochloric acid (ca 1%) or other inorganic acids (Wang et al. African J. Biotechnol. 10, 1460, 2011; CN 104387357-A, 2011 ). In this process to form aglycone (quercetin), the hydrolysed monosaccharides D-glucose and L-rhamnose are eliminated because the diglycoside rutinose bound by the β-glycosidic linkage to quercetin C-3 is completely cleaved (thus cannot be obtained). Furthermore, boiling in a strong acid can partially decompose all reactants, including quercetin, which deteriorates product quality and reduces yield. High-pressure hydrolysis with water alone (0.2 to 0.8 MPa) is also used for the hydrolysis of rutin, producing a mixture of quercetin and isocvercitrin and released monosaccharides (CN 1817876-A).

Enzymatic methods for the preparation of quercetin from rutin are also described in the academic and patent literature. E.g. Wang et al. (African J. Biotechnol. 10, 1460, 2011) tested the enzymes hesperidinase, snailase and cellulase-T2440, which are mixed preparations containing α-Lrhamnosidase, β-Dglucosidase, and other hydrolytic enzymes in varying proportions. The yields of quercetin were then 30 to 60%, producing partially deglycosylated products (eg isoquercitrin) and a mixture of monosaccharides (thus rutinosu could not be obtained). WO 2006/105843 A2 uses a variety of yeasts having glycosidase activity for deglycosylation. Of the released monosaccharides, glucose is utilized in situ by metabolizing yeast, leaving pure L-rhamnose (which yeast does not metabolize) and released quercetin. The disadvantage of the method is the low volumetric productivity and the presence of yeast in the final preparation, which complicates the isolation of the product (eg, simple filtration and washing cannot be used, since yeast are insoluble particles and remain in the preparation). CN 101787361-A utilizes an enzyme extracted from buckwheat seeds for deglycosylation of rutin, where the reaction is carried out in a mixture of water and ethanol (20 to 30%) and the enzyme is inactivated in the reaction. Nam et al. (Biotechnol. Lett. 34, 483, 2012) uses β-glucosidase from an extremophilic microorganism Pyrococcus furiosus to cleave rutin. The conversion of rutin was only in the presence of a cosolvent in a solution that was dimethylsulfoxide with a rutin concentration of 10 mM (ca 6 g / L). Although this enzyme is declared as a rutinosidase, monosaccharides are also produced in the hydrolysis of rutin, since the enzyme also has glucosidase and rhamnosidase activity alone, ie it is probably a mixture of different enzymes. It is practically impossible to obtain pure rutinium by this method.

A problem with existing processes is the very low solubility of rutin, which necessitates the use of either very low concentrations of reactants or the addition of organic solvents (e.g., ethanol, dimethylsulfoxide and others) to improve solubility (see above). Low substrate concentrations limit the bulk productivity of the process, and the addition of organic solvents also partially inhibits the enzymes used and reduces their stability. The prior art methods, which always work with dissolved routine, not only have very low volumetric productivity, but also do not allow

A3 or multiple uses of the enzyme, except for its possible immobilization, which, however, requires additional manufacturing costs, reduces the overall activity of the enzyme and the resulting quercetin, which is practically insoluble in water, is poorly removed from the immobilized enzyme. Organic co-solvents make it very difficult or practically impossible to obtain directly the rutinose which is in solution with them. In the case of using non-volatile solvents, there is no choice but to use column chromatography. Another is the problem of possible cosolvent residues in rutinose (but also in quercetin), which makes it impossible to use in cosmetics or even for oral administration.

The enzymes themselves for conversion can be obtained either commercially (e.g., naringinase, which is a mixture of α-Lrhamnosidase, β-D-glucosidase and other hydrolytic enzymes, from Penicillium decumbens) or by fermentation of the respective microorganisms. These enzymes can only be used once (inactivated to isolate the product) or in immobilized form, but this requires complete dissolution of the routine. Coarse buckwheat enzyme preparations are absolutely unsuitable for the parallel production of quercetin and rutinose. Complete dissolution of the routine is achieved by using a very low substrate concentration or by adding organic solvents that are volatile, flammable, toxic, increase process costs, and in addition denature the enzymes.

From the above described, it is clear that rutinium or quercetin cannot be isolated directly from the natural material in an economically advantageous manner. A suitable commercially available precursor of rutinose and quercetin is rutin. However, chemical hydrolysis of rutin is unsuitable due to the need to use aggressive chemicals and furthermore because rutinose is broken down into monosaccharides. Furthermore, the use of aggressive acid in production can have a negative commercial connotation - in contrast, the use of enzymes makes it possible to declare the resulting product as a biotechnologically prepared preparation (bio-product). The described enzymatic or other biological methods (yeasts) utilize high concentrations of cosolvents that reduce the activity of the enzyme used, lower bulk productivity, and the released rutinose is partially or completely cleaved into monosaccharides so that it cannot be obtained.

SUMMARY OF THE INVENTION

The disadvantages of the prior art methods of preparing rutinose and quercetin are overcome by the process of the present invention. It is an object of the present invention to provide a heterogeneous process for producing rutinose from rutin to produce quercetin as a valuable by-product, wherein the rutin in the form of a suspension, at a pH ranging from 2.05 to 10.1 and at a temperature of 10 to 70 ° C rutinosidase to produce rutinose and quercetin, followed by separation of the solid by-product quercetin from the reaction mixture by centrifugation, filtration or decantation. The rutinosidase enzyme is preferably 6-O-α-L-rhamnopyranosyl-3-D-glucopyranosidase. The routine, preferably at a concentration of 6-600 g / L, is suspended in an aqueous medium (for example, the aqueous medium may be water, a buffer or a buffered culture medium after submerged fermentation of a producing microorganism that produces the aforementioned rutinosidase enzyme). rutinosidase and its pH is greater than 2.05. The aqueous medium used preferably does not contain any organic solvents, in particular alcohols and DMSO. The enzymatic reaction then proceeds by cleaving rutin at a temperature above 10 ° C to rutinium (α-L-rhamnopyranoside- (1-> 6) D-glucopyranose) and quercetin (Scheme 1) without formation of any monosaccharides.

-4GB 2018 - 352 A3

quercetin

Scheme 1: Enzymatic deglycosylation of rutin according to the invention to produce rutinose and quercetin.

Quercetin, which is in the solid phase in the reaction mixture, is separated by centrifugation, filtration or decantation upon completion of the reaction. The reaction mixture, after all the rutin has been reacted and the solid quercetin has separated, contains the rutinium product which is obtained from the reaction solution by crystallization. The rutinose thus obtained is not contaminated with any monosaccharides, since under the above reaction conditions rutinose to monosaccharides is not hydrolyzed. Preferably, the rutinose is obtained as follows: the pH of the mother liquor is adjusted, preferably with lime water (saturated Ca (OH) ₂ ), to a pH of 7.0 to 8.0, then powdered charcoal (5 g / L) is added ) and diatomaceous earth (3 g / L) and the solution is brought to boiling, filtered, concentrated and allowed to crystallize (preferably at 0 to 5 ° C). Rutinosus can also be obtained from the solution by evaporation of water, preferably by lyophilization, the product being amorphous rutinose.

The remaining reaction solution with retained enzyme activity after removal of the solid quercetin can be used to re-produce rutinose (and quercetin) from rutin (after adjusting the pH to 2.05 or higher with a hydroxide solution, preferably potassium hydroxide), i.e. to suspend the rutin for another reaction. The rutinose content of the reaction mixture increases gradually as the reaction progresses, and upon completion of the reaction (for example, if enzyme activity is no longer sufficient), the solid quercetin is first removed and then rutinose is recovered from the mother liquor. The obtained rutinose and quercetin can be further purified by recrystallization.

An essential feature of the present invention is the solid state of the routine entering the reaction and the solid state of quercetin formed as a by-product of the reaction. Thus, the reaction mixture is a suspension of rutin in water, a buffer or a buffered culture medium after submerged fermentation of a production microorganism that produces the above-mentioned rutinosidase enzyme, and the reaction proceeds heterogeneously in suspension to form a quercetin suspension in a rutinose solution. The essential factor for the reaction is the solid phase of the rutin entering the reaction, as well as the production of quercetin in the solid phase, while the rutinose remains in solution. For solution reactions, the thermodynamic equilibrium applies, i.e. the reactant and the reaction product are in equilibrium. In contrast, the solid phase microenvironment reaction of the present invention, where the solid starting material rutin is converted to rutinium in solution and the solid by-product quercetin, instantly precipitates quercetin and thereby shifts the thermodynamic equilibrium towards the reaction products. The resulting solid quercetin does not participate in the thermodynamic equilibrium in the solution and thus cannot reverse its reaction, thus precipitating the insoluble quercetin results in the reaction of another rutin to rutinium and quercetin without feedback inhibition of the reaction by the product. The results are almost quantitative yields of rutinose and quercetin.

In contrast, in the presence of even a small amount of organic cosolvents in which rutin and especially quercetin is soluble, the product remains only partially in solution, the thermodynamic equilibrium shifts towards the reactant and thus blocks the reaction itself. Therefore, it is very important that the reaction system remains completely heterogeneous. Also in terms of product utilization, e.g.

The organic co-solvent, e.g. DMSO type, is completely contraindicated, merely because this solvent is virtually impossible to remove (e.g. by evaporation) from the mixture due to the high boiling point (189 ° C with simultaneous decomposition). Furthermore, DMSO has been shown to be toxic - for example, the developmental neurotoxin causes allergic reactions and has many other toxic reactions (Hanslick, JL; Lau, K; Noguchi, KK; Olney, JW; Zorumski, CF; Mennerick, S; Farber, NB) Dimethyl sulfoxide (DMSO) produces widespread apoptosis in the developing central nervous system (Neurobiology of disease. 34 (1): 1-10. Doi: 10.1016 / j.nbd.2008.11.006). Using even trace amounts of DMSO in the production of rutinose and quercetin would completely disqualify its use in human or animal nutrition for hygiene reasons. It would not even be possible to declare a commercially advantageous product quality as “bio” at all.

In a preferred embodiment, the concentration of rutin in the suspension at the start of the reaction is in the range of 6 g / L to 600 g / L, preferably in the range of 9 g / L to 450 g / L, most preferably 150 g / L to 250 g / L.

In one embodiment, the rutinosidase volume activity in the initial reaction mixture is in the range of 1.0 to 10 nkat / mL, preferably in the range of 2.5 to 7 nkat / mL, more preferably in the range of 2.5 to 3.3 nkat / mL mL.

In a preferred embodiment, the by-product quercetin obtained is purified by recrystallization, preferably by recrystallization from a NaOH solution of 50 to 500 mmol.L ¹ at a temperature in the range of from 20 to 100 ° C.

In a preferred embodiment, rutinose is obtained by evaporation of the reaction mixture after separation of the solids. More preferably, rutinose is obtained by lyophilizing the reaction mixture after separating the solids.

In another preferred embodiment, the rutinose is recovered from the reaction mixture after separation of the solids by adjusting the pH of the reaction mixture after separation of the solids (mother liquor), preferably with lime water (saturated Ca (OH) 2), to a pH of 7 0 to 8.0, then powdered charcoal (preferably about 5 g / L) and diatomaceous earth (preferably about 3 g / L) are added and the solution is brought to boiling, filtered, concentrated and allowed to crystallize (preferably at 0 to 5 ° C). The resulting rutinose crystals are separated by centrifugation, filtration or decantation.

In a preferred embodiment, the rutinosidase enzyme is 6-O-α-L-rhamnopyranosyl-3-D-glucopyranosidase.

In a more preferred embodiment, the 6-D-α-L-rhamnopyranosyl-3-D-glucopyranosidase is isolated from a microorganism of the genus Aspergillus, Penicillium, Mucor, Chaetomium, Acremonium or a bacterium of the genus Actinoplanes, preferably from a microorganism of the genus Aspergillus. Alternatively, 6-O- (R-L-rhamnopyranosyl-PD-glucopyranosidase may be prepared by heterologous expression in an industrial production microorganism, for example in Pichia pastoris yeast, preferably by heterologous expression of 6-OaL-rhamnopyranosyl-3-D-glucopyranosidase from Aspergillus niger in yeast Pichia pastoris Preferably 5% methanol is used as an inducer for heterologous expression (the resulting solution concentration is 0.5%).

In one embodiment, 6-D-α-L-rhamnopyranosyl-3-D-glucopyranosidase is prepared by submerged cultivation of the aforementioned microorganism, preferably Aspergillus niger, by submersive culture in a liquid medium containing rutin as an inducer.

In another embodiment, 6-D-α-L-rhamnopyranosyl-3-D-glucopyranosidase is prepared by heterologous expression in Pichia pastoris yeast in a liquid medium. Methanol at a concentration of 5% (final concentration in solution of 0.5%) is preferably used as an inducer for heterologous expression.

In a preferred embodiment, water and / or the reaction mixture containing the active enzyme rutinosidase remaining after separation of the quercetin from the reaction mixture and / or the filtered liquid medium is used to suspend the rutin in the first step according to the process for producing quercetin in a heterogeneous system.

-6C 2018 - 352 A3 after submerged cultivation of a 6-O- and -L-rhainnopyranosyl-β-gluk-glucopyranosidase producing microorganism. In all cases, the entire reaction system is heterogeneous.

The present invention claims an enzymatic process using completely unusual reaction conditions. It has been unexpectedly found that, under conditions in which most of the substrate and reaction product are present in the solid phase (suspension), the enzyme reaction proceeds in the solid phase microenvironment to form a solution of the rutinose product and precipitation of the by-product quercetin. Thus, it is a novel approach where the immobilized substrate (present in the heterogeneous reaction mixture as a solid phase) is treated with an enzyme in solution. Such an arrangement permits the use of very high substrate concentrations which are of an order of magnitude or more than those of the processes described so far (for comparison see CN 101787361-A and WO 2006/105843 A2), thereby increasing the bulk productivity of the process by an order of magnitude. In addition, this method completely eliminates the need for cosolvents, such as DMSO, which use other methods described, eg Nam et al. (Biotechnol. Lett. 34, 483, 2012), CN 101787361-A and WO 2006/105843 A2, and there is no need to eliminate them from the resulting product and hygiene problems with residual toxic cosolvents in the final product. Furthermore, this unusual but very efficient process of the present invention also allows easy separation of the very pure quercetin by-product from the enzyme used by simple filtration with washing directly on the filter or even by decantation, and the enzyme used in the reaction mixture can additionally preferably pH, preferably used again for the next reaction.

Thus, an essential advantage of the claimed process is that the enzymatic cleavage of rutin can also be carried out in a heterogeneous system where the starting rutin and the produced quercetin are both in suspension and the reaction itself takes place in a saturated solution or on an interfacial medium to produce an equimolar amount of rutinose. The reaction conditions are mild and rutinose is not cleaved to monosaccharides as in the prior art.

Another significant advantage of the claimed process is also that the enzyme preparation for the conversion of rutin to rutinium and quercetin can be directly the culture medium after fermentation of the production strain, preferably the microorganism Aspergillus niger, which has been selected by most screening as most suitable because it provides the highest rutinosidase activity. or even a heterologous production strain of Pichia pastoris, i.e. without the need for enzyme isolation and purification. The fermentation medium is only diluted with distilled water to the desired activity and the pH is adjusted to the desired value with an inorganic base or acid, preferably sodium hydroxide / phosphoric acid.

Another important advantage of the process of the present invention is the simple isolation of the product (rutinosy) which crystallizes from the reaction mixture after separation of the solids by centrifugation, filtration or decantation.

The post-reaction filtrate after the reaction contains cleaved rutinium which can be isolated and purified, for example, by crystallization. The filtrate from the enzyme reaction also contains still active rutinosidase so that it can be reused as an enzyme component after repeated pH adjustment, if necessary, to repeatedly convert rutin to rutin and quercetin. This process can be repeated, whereby the produced rutinose is removed from the industry by diluting the filtrate and further isolated. Isolation of rutinose, which has the potential to be used in cosmetics, is very simple due to the fact that no other glycosidases are present in the process of the present invention, nor do such conditions (e.g., very low pH and high temperature) at which to break it down to monosaccharides. As a result, only rutinose and traces of inorganic salts from the medium are readily removed, such as phosphates by neutralization with lime water and crystallization of rutinose from the concentrated solution.

The preparation of high purity rutinose and quercetin is accomplished by recrystallization of the crude product after enzyme conversion. Recrystallization of rutinose has been described above. Recrystallization of quercetin is performed by dissolving the crude quercetin at boiling in a 0.1 to 1 NaOH solution.

-7 CZ 2018-352 A3 mol.L ¹ pH heat brought to pH 9 to 10 for cooling and crystallization proceeds. The resulting product, having a content of over 99% by weight of quercetin, is washed with dilute sulfuric acid at a concentration of 0.05 mol / ^L and then three times with water. The recrystallization can be repeated, further increasing the purity of the product.

The fermentation is carried out sterile under aerobic conditions, with stirring, and preferably at a temperature of 28 ° C in shake flasks or in a stirred fermenter. After fermentation, which typically lasts 4 to 7 days, the mycelium of the microorganism is removed by centrifugation or filtration. The remaining rutinosidase-containing medium, after adjusting the pH to at least 2.05 and warming to a reaction temperature in the range of 10 to 60 ° C, preferably 40 ° C, is used directly for the reaction, i.e. the routine is stirred in and the reaction is stirred. it is allowed to proceed to the desired conversion under constant conditions and with stirring. The protein sequence, i.e. the rutinosidase from Aspergillus niger, is known and this enzyme was cloned in a standard manner in the expression microorganism Pichia pastoris (Simcikova et al. Adv. Synth. Catal. 357, 107, 2015).

Clarification of drawings

Giant. 1 HPLC chromatogram documenting the course of enzymatic cleavage of rutin. Chromolith Performance RP-18e column, 100x 3 mm (Gauge); Chromolith RP-18e precolumn, 5x4.6 mm; mobile phase [v / v]: A = 5% acetonitrile, 0.1% formic acid, B = 80% acetonitrile, 0.1% formic acid, in water (v / v); gradient elution 0 to 3 min 7 to 25% B, 3 to 5 min 30% B, 5 to 7 min 7% B; flow rate 1.5 mL / min, 25 ° C, detection at 360 nm. A - 0 hours (start of reaction), B - 4 hours, C - 7 hours (end of reaction). 1- Rutin, 2-quercetin. The x-axis shows the UV detector response in mV, the y-axis the time in minutes.

Giant. 2 Rutin to rutinium conversion process, input conc. rutin 200 g / L, reaction volume 10 mL, 40 ° C, pH 3, rutinosidase containing 4.8 nkat / mL medium.

Giant. 3 Conversion of rutin to rutinium using different amounts of enzyme, input conc. rutin 200 g / L, reaction volume 3 mL, 35 ° C, pH 3.5.

Giant. 4 Conversion of rutin to rutinium at different pH, input conc. rutin 200 g / L, reaction volume 3 mL, 35 ° C, rutinosidase amount of 1.11 nkat / mL.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Preparation of rutinosidase enzyme by Aspergillus niger

The enzyme rutinosidase (EC 3.2.1.168) was prepared by submerged fermentation of Aspergillus niger in liquid medium. The production fermentation medium had the following composition [g / L]: 5.0 routines; 15.0 KH2PO4; 4.0 NH4Cl; 0.5 KCl; 5.0 yeast extract; 1.0 casein hydrolyzate; 1 mL of trace element solution, pH adjusted to 5.0; After autoclaving at 121 ° C and for 30 minutes, 1.0 mL of sterile 10% (w / v) MgSCL./ILO solution was added. The trace element solution had the following composition [g / L]: 50.00 EDTA (ethylenediaminetetraacetic acid); 22.00 ZnSO ₄ · 7 H2O; 5.54 CaCL; 5.06 MnCL · 4 H ₂ O; 4.99 FeSO ₄ · 7 H ₂ O; 1.10 (NH ₄ ) 6 Mo ₇ O ₂ 4 · 4 H ₂ O; 1.57 CuSO ₄ · 5 H ₂ O and 1.61 CO ₂ · 6 H ₂ O, the components were dissolved in distilled water and the pH was adjusted to 6.0 with a 40% KOH solution. For culture of microorganism grown on slant agar [g / L]: 125 glycerol, 45 malt extract, 15 NaCl, 0.44 NH4NO3, 0.06 MgSO4. 7 H2O 0.0015 CuSCU. 5 H2O, 30 agar; pH 5.5 (HCl) was sterile pipetted with 6 mL of a 0.1% Tween 80 solution (polyoxyethylene sorbitan monooleate) and by scraping the spores of the Aspergillus niger microorganism into the solution, a spore suspension and a portion of the mycelia were formed. Up to 200 mL of inoculation medium was

2018 - 352 A3 inoculated with 1 mL of this suspension (10 ⁷ spores / mL). The inoculated inoculation medium was cultured in a 500 mL Erlenmayer flask at 28 ° C for 6 days with stirring (200 rpm). On the sixth day after inoculation of the culture medium, the mycelium was separated by filtration. The rutinosidase volume activity at the end of the fermentation was 0.58 nkat / mL.

Example 2: Preparation of rutinosidase enzyme by Pichia pastoris

Preferably, the rutinosidase (EC 3.2.1.168) can also be prepared by heterologous expression in, for example, Pichia pastoris yeast according to the following example. Heterologous expression of rutinosidase in Pichia pastoris KM71H yeast was continued for 5 days. An expression vector containing the rutinosidase gene (pPicZaA-Rut; Simcikova et al. Adv. Synth. Catal. 357, 107, 2015), bearing resistance to the antibiotic zeocin) was first linearized and subsequently electroporated into P. pastoris KM71H cells. The transformed cells were cultured on YPD (Yeast Peptone Dextrose medium) on Petri dishes (YPD [g / LJ: 10.00 yeast extract; 20 peptone; 20 glucose, 15 agar and zeocin antibiotic (final concentration 1 mg / mL)). The plates were incubated for 3 days at 28 DEG C. The grown colonies were then used for liquid culture and screened to find the colony producing rutinosidase as large as possible according to the Easy Select Pichia Expression Kit (Invitrogen, USA). The selected rutinosidase-producing colony was cultured overnight in liquid YPD medium, the next day the pellet was separated by centrifugation and then dissolved in a liquid YPD medium containing 15% glycerol and the preserved cans of rutinosidase-producing cells (100 µL) were stored at -80 °. C. The production of rutinosidase was performed in 1 L medium having the following composition: 700 mL water, 100 mL 10% glycerol 100 mL of 1 M potassium phosphate buffer pH 6; 100 mL of 13.4% YNB (Yeast Nitrogen Base, ROTH) and 2 mL of a 0.2% biotin solution. Cryopreserved transformed P. pastoris cells containing 100 µL cells were added to 1 L medium, and the seeded medium was cultured in a 3 L Erlenmeyer flask at 28 ° C for one day with stirring (250 rpm). On the second day, the cells separated by centrifugation were resuspended in 200 mL of medium in a 1 L Erlenmayer flask containing 140 mL of water; 20 mL of 1 M potassium phosphate buffer pH 6; 20 mL 13.4% YNB (Yeast Nitrogen Base, ROTH); 400 µL of a 0.2% biotin solution and 20 mL of 5% methanol in ultrafiltration sterilized water. The cultivation was carried out for 4 days at 28 ° C with stirring (250 rpm). Gene expression was induced every day by the addition of 1 mL of 100% methanol for 4 days. The resulting rutinosidase-containing supernatant was separated from the cells by centrifugation. The volume activity of rutinosidase at the end of the fermentation was typically about 7 nkat / mL.

The supernatant after the end of the fermentation was dialyzed for purification (dialysis intestine 33 mm wide, cut off 10 kDa, Sigma-Aldrich) for 2 h against 10 mM acetate buffer pH 3.5. Subsequently, the supernatant was diluted 2 times with nano-water and the pH was adjusted to pH 3.5 with acetic acid. Ion exchange chromatography was performed on a Fractogel EMD SO3 column (1.5 x 10.0 cm, Gauge). The loading buffer A (10 mM acetate buffer, pH 3.5) and elution buffer B (containing additionally 1M NaCl) were used as mobile phase. The mobile phase flow rate was 2 mL / min and pure rutinosidase was eluted with 100% buffer B. The eluted protein was concentrated by ultrafiltration on an Amicon 8400 apparatus (Merck-Millipore). The ultrafiltration membrane had a eut-off of 10 kDa. The yield of this purification is about 30%.

The resulting rutinosidase activity in the reaction medium should preferably have a value of 2 to 2.8 nkat / mL for optimal reaction (i.e., a conversion time of about 7 h). The enzyme activity of rutinosidase was measured spectrophotometrically by a discontinuous method using a chromogenic substrate of pnitrophenyl-rutinoside (pNP-Rut). The absorbance of p-nitrophenol was measured at 420 nm. The reaction mixture containing 10 µL rutinosidase solution, 10 µL pNP-Rut (10 mM) and 30 µL citrate phosphate buffer (pH 5) was incubated in a thermomixer for 10 minutes at 35 ° C (600 rpm). The enzyme reaction was terminated by the addition of 1 mL of Na ₂ CO 3 (0.1 M).

-9GB 2018 - 352 A3

Example 3: Preparation of rutinose and quercetin by cleavage of rutin

Rutinosus can be prepared by enzymatic cleavage of rutin to form rutinose and quercetin according to the following example. 80 mL of culture medium containing rutinosidase obtained by heterologous expression in P. pastoris cells according to Example 2, whose pH was adjusted to pH 3.0 with phosphoric acid (Fig. 4), was diluted 2.5 times with nano-duct to a total volume of 200 mL, the rutinosidase activity is preferably in the range of 2.5 to 3.3 nkat / mL (Fig. 3). Enzyme activities of 1.0 were also tested; 2.0; At 5.0 and 10.0 nkat / mL, the reaction was satisfactory. Even at concentrations below 1 nkat / mL, the reaction proceeds, but the conversion is incomplete. To the thus prepared rutinosidase solution was added 40 g of rutin (final conc. 200 g / L) and the reaction mixture was further incubated at 40 ° C, preferably for 7 h (Fig. 2) with stirring. The progress of the reaction was monitored by HPLC, under the following conditions: Chromolith Performance RP-18e, 100 x 3 mm (Gauges); Chromolith RP-18e precolumn, 5 x 4.6 mm; mobile phase: A = 5% acetonitrile, 0.1% formic acid, B = 80% acetonitrile, 0.1% formic acid, in water (v / v); gradient elution 0 to 3 min 7 to 25% B, 3 to 5 min 30% B, 5 to 7 min 7% B; flow rate 1.5 mL / min, 25 ° C, UV detection at 360nm, room temperature (see Figure 1). The reaction was quenched by separating the solid and liquid phases by filtration. The filter cake was washed with hot water (3 x 10 mL water) to remove impurities. The filter cake containing almost pure quercetin was then dried in an oven at 50 ° C.

The obtained fine yellow powder (19.2 g; 97%) contained 98% quercetin; 0.4% of rutin and ca 1.6% of unidentified impurities (all percentages by weight). Conversion of rutin while maintaining the reaction conditions (temperature, pH, and bulk activity of the enzyme) does not depend on the reaction volume (volumes of 0.5 L to 10 L were tested). According to the above procedure, the reaction was performed at an initial rutin concentration of 6 g / L to 600 g / L (g / L: 6.4; 128; 256; 320; 450; 600). At higher concentrations exceeding 150 g / L of the starting routine, it was necessary to extend the reaction time to 20 to 60 hours to achieve a corresponding, i.e. at least 98%, conversion by analytical HPLC as described above. The structure of quercetin was confirmed by NMR II and ¹³ C (in CD3OD) and by mass spectrometry by HPLC MS and by comparison with an authentic quercetin standard (Sigma). The purity of the product was determined by HPLC as described above (see Fig. 1).

Example 4: Isolation of rutinose from the reaction mixture

In order to obtain rutinose, the remaining filtrate after further separation of quercetin was further processed preferably by the following procedure. The pH of the solution was adjusted to pH 7.0 to 8.0 with lime water (aqueous saturated Ca (OH) ₂ ), then powdered charcoal (5 g / L) and diatomaceous earth (3 g / L, Gauge) were added and the solution was brought to reflux, filtered and concentrated on a evaporator. The rutinose sample thus concentrated was heated to boiling and, after cooling, the sample was allowed to crystallize in the refrigerator. In order to obtain rutinose, crystallization can be bypassed and the resulting solution can only be evaporated under vacuum on a rotary evaporator or preferably also by lyophilization.

Example 5: Preparation of rutinose and quercetin using the reaction medium again

The reaction medium used (100 mL) after rutin conversion and quercetin separation according to Example 3 can be reused to cleave rutin into rutinium and quercetin since most of the rutinosidase enzyme activity is retained in solution. After checking the pH and possibly adjusting it to pH 3, the solid was routinely added to the medium to a final concentration of 200 g / L. The procedure was as in Example 3, except that the reaction time had to be increased to 18 hours (according to the conversion monitored by HPLC). The yield and purity of the quercetin obtained were the same as in Example 3. Such recycling of the enzyme can be performed several times depending on the residual concentration of the enzyme. Rutinose accumulates in the resulting filtrate and can be obtained as described in Example 4.

- 10GB 2018 - 352 A3

Example 6: Recrystallization of quercetin

Quercetin having a purity greater than 99% by weight was obtained by repeated crystallization of the product obtained according to Example 3. The yellow powder (140 g, 98% by weight of quercetin) was stirred in 1 liter of 0.25 M NaOH. The mixture was brought to reflux while stirring to dissolve the quercetin and then the hot solution was filtered. Slow cooling (20 h at room temperature followed by 4 h at 5 ° C) followed. The precipitated yellow quercetin crystals were filtered off, washed with water at 40 ° C (500 mL) and dissolved in 100 mL of 1 M NaOH. Slow cooling (20 h at 25 ° C) followed by cooling (4 h at 5 ° C). The precipitated crystals were filtered off and resuspended in water. The aqueous suspension was acidified with dilute sulfuric acid to pH 3.5 and the insoluble material was filtered off and dried; in this operation, alkali residues (sodium ions) from the previous crystallization were separated from the quercetin suspension. The obtained yellow powder (6 g) contained 99.85% by weight of quercetin and 0.15% by weight of rutin as determined by HPLC analysis according to Example 3. All impurities were thus removed. Lower purity quercetin can be obtained by further precipitation from the mother liquors, in particular by further standing in the cold and / or by acidification.

Example 7: Recrystallization of rutinose

The rutinose obtained according to Example 4 is dissolved in distilled water to a concentration of 200-400 g / L at elevated temperature. If the solution is colored (eg yellow), it is bleached by the addition of activated carbon (1 g / L) and diatomaceous earth (1 to 3 g / L) with short boiling and hot filtration. The clear solution obtained is allowed to cool and then crystallized overnight at a lower temperature, preferably 3 to 7 ° C. Preferably, the solution to be crystallized can be seeded with a small amount of rutinose crystals. The crystals are separated by filtration or centrifugation. They are only aspirated on the filter and dried without washing. After evaporation, the mother liquor yielded additional rutinose crystals. Recrystallization yields are between 70 and 85%. Recrystallized rutinose is a white or slightly yellowish fine crystalline powder which is hygroscopic and must be kept in a tightly closed container.

Industrial applicability

Rutinose has a potential use in cosmetics and as a low-calorie sweetener, the by-product quercetin is a potent antioxidant with high chemoprotective potential and is useful in nutraceuticals, functional foods, to improve the nutritional value of foods and, for example, in cosmetics.

PATENT CLAIMS

Claims (10)

Process for the production of rutinose from rutin in a heterogeneous system, characterized in that rutin in the form of a suspension in an aqueous medium at a pH in the range of 2.05 to 10.1 and at a temperature of 10 to 70 ° C is subjected to rutinosidase enzyme to form rutinose and quercetin, followed by separation of quercetin from the reaction mixture by centrifugation, filtration or decantation, and rutinose being obtained by crystallization or evaporation of the reaction mixture after separation of the solids. The method of claim 1, wherein the aqueous medium is water, a buffer, or a buffered culture medium after submerged fermentation of a producing microorganism that produces a rutinosidase enzyme. Method according to claim 1 or 2, characterized in that the concentration of rutin in the suspension at the start of the reaction is in the range from 6 g / L to 600 g / L, preferably from 9 g / L to 450 g / L, more preferably from 150 g / L to 250 g / L. - 11 GB 2018 - 352 A3 Method according to claim 1, 2 or 3, characterized in that the rutinosidase volume activity in the initial reaction mixture is in the range of 1.0 to 10 nkat / mL, preferably in the range of 2.5 to 7 nkat / mL, more preferably in the range of from 2.5 to 3.3 nkat / mL. The method of any one of the preceding claims, wherein the rutinosidase enzyme is δ-O-α-L-rhamnopyranosyl-β-D-glucopyranosidase. The method of claim 5, wherein the β-O-α-L-rhamnopyranosyl-β-D-glucopyranosidase is isolated from a microorganism of the genus Aspergillus, Penicillium, Mucor, Chaetomium, Acremonium or Actinoplanes; or is prepared by heterologous expression in an industrial producing microorganism, preferably β-O-α-L-rhamnopyranosyl-β-D-glucopyranosidase from a microorganism of the genus Aspergillus, most preferably from Aspergillus niger, prepared by heterologous expression in yeast Pichia pastoris. 7. The method of claim 6, wherein the .beta.-O-.alpha.-L-rhamnopyranosyl-.beta.-D-glucopyranosidase is prepared by submerged culture of a microorganism in a liquid medium. The method according to claim 6, characterized in that 6-O-α-L-rhamnopyranosyl-β-D-glucopyranosidase is prepared by heterologous expression in Pichia pastoris yeast in a liquid medium. Process according to any one of the preceding claims, characterized in that the pH of the reaction mixture is adjusted to a pH of 7.0 to 8.0 after completion of the reaction and after the solids have been separated off, followed by the addition of powdered activated carbon and diatomaceous earth to the solution. the mixture is boiled, filtered, concentrated and crystallized to give crystalline rutinose. Process according to any one of the preceding claims, characterized in that the quercetin formed is purified by recrystallization, preferably by recrystallization from a NaOH solution having a concentration of 50 to 500 mmol.L ¹ at a temperature ranging from 20 to 100 ° C.