JPH08282A - Production of 5-methyluridine - Google Patents

Abstract

PURPOSE:To produce 5-methyluridine useful e.g. as a synthetic intermediate for azidothymidine, etc., in high efficiency, purity and yield by culturing a specific microorganism, etc., and treating with a ribosephosphate, etc. CONSTITUTION:This 5-methyluridine is produced by culturing a microbial strain capable of forming 5-methyluridine from a ribose-1-phosphate(salt) and 5- methyluracil [e.g. Arthrobacter simplex (FERM P-10068)] or a microbial strain capable of forming 5-methyluridine from a nucleoside, an inorganic phosphoric acid(salt) and 5-methyluracil, removing a part or total of the medium component, treating the microbial cells with a ribose-1-phosphate(salt) and 5-methyluracil or a nucleoside, an inorganic phosphoric acid(salt) and 5-methyluracil and crystallizing and separating the produced 5-methyluridine.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing 5-methyluridine, which is useful as a raw material for pharmaceuticals. 5-methyluridine is azidothymidine, which is commercially available as an AIDS drug, and d4T (2 ', 3'-didehydroxy-2', 3'-dideoxythy) which is also in clinical trials.
midine) is seen as a promising intermediate.

[0002]

2. Description of the Related Art 5-Methyluridine has hitherto been produced by a chemical synthesis method, and its purification was mainly carried out by crystallization using an organic solvent such as methanol or ethanol. (J. Am. Chem. Soc., 78, 2117 (1956); JP-A-63-63668; Helvetica Chimica Acta, 2179 (19).
80); Synthesis, 259 (1982))

These techniques are not useful as a method for producing 5-methyluridine, including purification, because impurities produced as by-products differ from those obtained by converting nucleic acids as raw materials and using microorganisms.

As a method for producing 5-methyluridine utilizing a microorganism, there is known a method for producing 5-methyluridine by allowing 5-methyluracil to act on nucleoside or ribose-1-phosphate in the presence of the microorganism. (JP-A-2-23882)

However, this patent does not have a specific description of a method for purifying 5-methyluridine, which is insufficient for producing 5-methyluridine as a product.

In particular, obtaining large crystals of 5-methyluridine by crystallization can improve the separability of the crystals and reduce the scale of the apparatus required for the separation, and the separation from impurities is also good. However, it is indispensable for obtaining a high-purity product, but there is no description about it.

Usually, the degree of supersaturation and impurities have a great influence on the crystal growth, and above all, the composition of the impurities is completely different depending on the system to be crystallized, and this control is an important key for determining whether crystallization is possible or not. In many cases, characteristic control in a given system is necessary.

Therefore, in the case of 5-methyluridine utilizing microorganisms, the information necessary for biochemical production is known, but it was necessary to independently develop a method for purification from the reaction solution.

When 5-methyluridine was crystallized from the reaction solution by some pretreatment and concentration, only crystals having a size of 20 to 30 micrometers (μm) were obtained, which resulted in a very high separation rate. Slowly, it turned out that the separator was a big one. Further, it has been found that this size is completely unsuitable for the separation of impurities by utilizing the difference in crystal grain size described later.

5-Methyluridine can be produced by enzymatic reaction using a nucleic acid as a raw material using a microorganism. At that time, in the reaction product, nucleic acid which is an unreacted product and nucleic acid which is a by-product. Species are included as impurities. When 5-methyluracil (thymine) and guanosine are used as raw materials for producing 5-methyluridine,
Impurities in the reaction solution were mainly thymine, guanine, guanosine, and 2-Amino-7-β-D-ribofuranos.
There is yl-7H-purine-6 (1H) one (sudoguanosine). Among them, for example, thymine has a solubility change pattern with respect to temperature, pH and the like that is similar to that of 5-methyluridine, and thus it is difficult to remove it by crystallization. Also,
Since the ion dissociation patterns are similar, it is difficult to remove them by treatment with an ion exchange resin or the like.

These nucleic acid-based impurities are peculiar to the method of production by a microorganism using nucleic acid as a raw material, and public knowledge cannot be utilized. Moreover, a method for separating these is not described in the known literature.

[0012]

[Problems to be Solved by the Invention] In order to efficiently produce 5-methyluridine and to obtain a highly pure product, the grain size of 5-methyluridine crystals from a reaction solution using a microorganism is increased to form crystals. Increase the efficiency of separating mother liquor,
A method for improving the separation efficiency between this crystal and other impurity crystals will be developed.

[0013]

Means for Solving the Problems The inventor has cultured a microorganism, produced 5-methyluridine by an enzymatic reaction using nucleic acids as a raw material, and studied the crystallization and separation method of the microorganism when the culture solution of the microorganism was examined. The present invention has been completed by finding that there is a large difference in the grain size of crystals obtained by the crystallization of 5-methyluridine in the subsequent step depending on the treatment method of. That is, the present invention is
Microorganism capable of producing 5-methyluridine from ribose-1-phosphate or a salt thereof and 5-methyluracil, or microorganism capable of producing 5-methyluridine from a nucleoside, infinite phosphoric acid or a salt thereof and 5-methyluracil In the method for producing 5-methyluridine by enzymatic reaction using, after culturing the microorganism, (a) removing a part or all of the medium components, and (b) adding ribose-1 to the microorganism. -Activating phosphoric acid or a salt thereof and 5-methyluracil or nucleoside, infinite phosphoric acid or a salt thereof and 5-methyluracil to produce 5-methyluridine, and then (c)
The present invention relates to a method for producing 5-methyluridine, which comprises crystallizing the produced 5-methyluridine and separating it.

The microorganism used in the present invention is ribose-
5 from 1-phosphoric acid or its salt and 5-methyluracil
-A microorganism capable of producing methyluridine or a microorganism capable of producing 5-methyluridine from nucleoside, infinite phosphoric acid or a salt thereof and 5-methyluracil can be used. For example, Achromobacter, Acinetobacter, Aeromonas, etc.
The microorganisms described in JP-A-2-23882 can be mentioned. Examples of particularly preferable microorganisms include microorganisms belonging to the genera Arthrobacter, Cellulomonas, Flavobacterium, Klebguera, Microbacterium, Micrococcus, and Sarcinia. Specific examples thereof include the following microorganisms. Arthrobacter simplex FERM P-10
068 Cellulomonas flavigena ATCC 486 Flavobacterium rhenanum FERM BP-
1862 Klebsiella pneumoniae ATCC 8308 Microbacterium lacticum ATCC 818
0 Micrococcus luteus FERM P-7399 Sarcina lutea FERM P-7400

Cultivation of the above-mentioned microorganism is carried out by using a carbon source, a nitrogen source,
Using an ordinary medium containing inorganic ions such as P, S, Fe, and Mn, and if necessary, micronutrients such as vitamins or proteolytic products, organic nitrogen sources such as yeast extract, and performing the usual culture method. Good.

As a method for removing the medium components from the obtained culture solution of the microorganisms, the supernatant is removed after the culture solution is naturally precipitated,
A usual method such as centrifugation or filtration can be used. By these methods, the cells and mother liquor can be easily and arbitrarily divided. The ratio of the amount of the medium components removed is preferably 50 to 99% by volume of the culture solution, and particularly preferably 7%.
It is 0 to 90% by volume.

After removing a part or most of the medium components from the culture solution, the reaction substrate is directly added to the microorganism-containing solution, or the microorganism-containing solution is diluted with a buffer such as an inorganic phosphate buffer or Tris buffer. Risose-1-phosphate or a salt thereof and 5-methyluracil, or nucleoside, infinite phosphoric acid or a salt thereof and 5-methyluracil are added to carry out an enzymatic reaction to produce 5-methyluridine. The reaction has a pH range of 4 to 10, preferably 6 to 8,
The temperature range is 20 to 70 ° C., preferably 50 to 70 ° C., and it is carried out for 10 minutes to 10 days while standing or stirring.

When the substrate of nucleic acids was added to the culture medium as it was without reacting with the medium components of the culture medium and the reaction was carried out, the grain size of the obtained crystals was 20 to 30 μm in major axis. It was found that with this particle size, the separation speed of crystals, for example, when centrifugal filtration is very slow, and a large apparatus is required.

On the other hand, when a part or most of the medium component of the culture solution is removed and then a substrate of nucleic acids is added and the reaction is carried out, crystals having a particle size of 50 to 550 μm are obtained. I found a thing. With this particle size, the separation speed of the crystal is increased, and the particle size can be separated from other impurity nucleic acids described later.

When the inventor studied the purification of 5-methyluridine, as described above, when a part or most of the medium components were removed from the culture solution and then a substrate was added and reacted, it was obtained in a later step. It was discovered that the crystals of 5-methyluridine were relatively large, and the idea that this property could be utilized for purification of 5-methyluridine was obtained. Generally speaking, when trying to obtain crystals with uniform grain size industrially, the fluid to be crystallized is caused to flow from below by itself, and large crystals are classified into the lower part and fine crystals into the upper part, and the upper part is classified. Although a method of dissolving and recirculating fine crystals of the above is taken out of the system, as in the present invention, there are few methods of separating the product crystal and the impurity crystal by the sedimentation speed. The reason is that there is no system with such a particle size distribution, impurity crystals are incorporated into the product crystals, and even if they can be separated, the purity does not rise, and it is possible to classify them. It is thought that the idea does not reach that point even if the quality is grainy or crystalline.

In the case of 5-methylurdine, the crystal grain size of the commercially available reagent was about 50 μm at the most, but when the inventor recrystallized it from water, crystals of about 500 μm were obtained.

On the other hand, 5 obtained from an actual liquid system containing impurities
-As a result of powder X-ray diffraction of methyluridine crystals, 5-methyluridine does not form a "mixed crystal" with other impurities, and if only crystals of 5-methyluridine can be taken out from the mixed crystal group, We have found that we can obtain pure products. In addition, the crystal grain system of 5-methyluridine in an actual liquid system is usually 300 to 600 μm, and even if it is small, it is 50
To 100 μm, and the grain size on the impurity side was 5 to 50 μm. Furthermore, by observing the crystallization slurry that was left standing after stirring, it was found that the 5-methyluridine crystals were in the lower part of the glass container, and the liquid in the upper part was cloudy for a short time. Then, I got the idea that other crystals could be separated.

As a concrete method for separating the crystals at a sedimentation rate, there is a decantation method. This is a method in which the crystal slurry is mixed well and then allowed to stand, and while 5-methyluridine largely precipitates and other crystals are suspended, the layers other than 5-methyluridine are tilted away and tilted away. Crystallize the effluent, return the mother liquor to its original container,
A 5-methyluridine crystal free from impurity crystals can be obtained by repeating the above-mentioned operation again several times. Moreover, with a large device, it is difficult to tilt the container, so that it is necessary to rely on mechanical force. For example, a method of sucking out the non-sedimentation layer using a pump, or providing an overflow port at an appropriate position and allowing the upper liquid to overflow after sedimentation can be considered. Further, a general fluidized bed type continuous crystallization tank is improved, a method of flowing a clarified mother liquor from the lower part of the tank, causing no flow of 5-methyluridine crystals, and flowing out only impurity crystals, or a so-called super decanter, A method of separating large crystals and small crystals at a stretch with a centrifuge capable of classifying and separating crystals such as hydrocyclone can be considered.

By repeating decantation or mechanical separation using the above centrifugal separator or the like, it is possible to obtain a predetermined separation efficiency and a predetermined product purity.

In any of these methods, the fluid containing the separated impurity crystals is crystal-separated by a centrifuge, filtration or the like,
If the mother liquor is returned to the original apparatus, the impurity crystal separation efficiency can be further increased.

The separated crystal layer on the 5-methyluridine side is separated by centrifugation, filtration or the like, and at this time, the crystal is washed with water, if possible with cold water, to obtain a crystal with higher purity. .

In producing 5-methyluridine,
A typical example of a method of converting a nucleic acid as a raw material using a microorganism is a method in which nucleoside, inorganic phosphate and 5-methyluracil (thymine) are allowed to act in the presence of the microorganism. Here, examples of the nucleoside include adenosine, guanosine, inosine, uridine, cytidine or xanthosine (JP-A-2-23882). In this reaction, each residual nucleoside as an impurity,
There is 5-methyluracil (thymine), a base by-produced from the nucleoside used and phosphoric acid. It is easy to separate these impurities by the difference in solubility with 5-methyluridine, but the patterns of change in solubility of nucleic acid components with respect to temperature and pH are similar to each other, and further separation is not easy. Absent.

In the process of examining removal of impurity nucleic acids in this reaction solution, the inventor found that crystals of 5-methyluridine were relatively large, whereas other crystals were not. Furthermore, it was discovered that the sedimentation rate of 5-methyluridine in the fluid was significantly higher than that of other crystals.

In addition to the conditions of JP-A-2-23882, after removing a part or most of the medium components from the culture solution and adding a substrate for reaction, the reaction solution is cooled, filtered, decolorized with activated carbon and filtered. The obtained filtrate was concentrated, cooled and crystallized in the range of 3 to 15 ° C., and among the crystals precipitated without adding any other solvent, the average grain size of 5-methyluridine crystals was 50 to 550 μm. While that of impurities is 1
It was about 0 to 30 μm.

Therefore, after the 5-methyluridine crystals in the fluid are allowed to settle for 5 to 10 minutes, the portion other than the 5-methyluridine crystal layer is tilted out of the container (decantation), and the effluent is further discharged. After separating the crystals inside, the mother liquor was returned to the container, and after stirring, the operation of decanting was repeated, whereby high-purity 5-methyluridine containing little other crystals was obtained.

Depending on the size of the crystallizer or the grain size, 5
-Although the time required for the sedimentation of methyluridine varies, the required time at that time is to extract a part of the crystal mud and allow it to actually settle in a graduated cylinder, etc., and measure the time and the settling distance required for it. The sedimentation time can be easily set by calculating the desired sedimentation velocity in the device.

On the other hand, since the crystal grain size of impurities is almost constant at all times, it is sufficient to consider the sedimentation time only on the 5-methyluridine side. If the sedimentation time is taken for too long, the impurity crystals will also sediment to some extent. With a small device such as a graduated cylinder, a sedimentation layer of fine crystals was formed in about 30 minutes. Also in this case, the time for the impurity crystals to settle is
It can be easily calculated based on the scale of the device and the settling time limit can be determined thereby.

When a fluidized bed type continuous crystallization tank is improved or a fluidized bed classification type crystallizer is designed and used, the refining mother liquor is caused to flow from the lower portion of the tank, and crystals of 5-methyluridine are not allowed to flow out. By measuring the linear velocity of the fluid required to flow out only the impurity crystals, the linear velocity in the actual device can be easily determined.

Similarly, when a so-called super decanter is used, the feed flow rate in the actual machine can be easily set by measuring the fluid feed flow rate per settling area and the crystal separability using a small machine. .

Here, we have found that the method utilizing the difference in the sedimentation rate of crystals is an effective method for separating 5-methyluridine from other nucleic acid components.

The above-mentioned method exhibited a particularly great effect on the separation of thymine, which is extremely difficult to separate from 5-methyluridine. Since thymine has a solubility change pattern similar to that of 5-methyluridine, which is difficult to separate, and also has a similar ionic dissociation pattern, which is difficult to separate even with an ion exchange resin, etc. The method method of the present invention was very effective.

Further, other coexisting nucleic acids such as guanosine, guanine, and pseudoguanosine were also very effective separation means.

The reason why crystals of 5-methyluridine having a large particle size can be obtained only when a part or most of the medium components in the culture medium of the microorganism are removed is not clear. Amino acids and other ingredients contained in
For example, it is considered that sugar reacts with the product and inhibits the growth of 5-methyluridine crystals. The temperature of the reaction using the present microorganism is 60 ° C., which is higher than that in the production using general microorganisms, and it is considered that this promotes the above-described byproduct reaction. It is believed that this has led to the need for removal of components.

[0039]

The present invention will be further described with reference to the following examples.

Example 1 In accordance with Example 1 of JP-A-2-23882, Flavobacterium rhenanum FE
RM BP-1862 was cultured to prepare a cell culture solution. The cell culture solution was centrifuged to remove 50% of the medium components. To this, add Tris buffer to restore the original volume, and
Methyluracil and guanosine were added, and the temperature was kept at 60 ° C. for reaction. The reaction solution is cooled, filtered, decolorized with activated carbon,
It was filtered, concentrated in vacuo and cooled to 5 ° C. The crystal size in the obtained crystallization slurry was about 80 μm. In addition, 5-methyluridine and other impurity nucleic acid crystals were well separated by spontaneous precipitation and separated into two layers.

Example 2 According to Example 1 of JP-A-2-23882, Flavobacterium rhenanum FE
RM BP-1862 was cultured to prepare a cell culture solution. This cell culture solution was centrifuged to remove 90% of the medium components. To this, add Tris buffer to restore the original volume, and
Methyluracil and guanosine were added, and the reaction and post-treatment were carried out in the same manner as in Example 1 of the present invention. The crystal size in the obtained crystallization slurry was about 70 μm. Also, 5-
Methyluridine and other impurity nucleic acid crystals were well separated by spontaneous precipitation and separated into two layers.

Example 3 Flavobacterium rhenanum FE according to Example 1 of JP-A-2-23882
RM BP-1862 was cultured to prepare a cell culture solution. This cell culture solution was centrifuged to remove 90% of the medium components. To this, an inorganic phosphate buffer was added to restore the original volume, the substrates 5-methyluracil and guanosine were added, and the reaction and post-treatment were carried out in the same manner as in Example 1 of the present invention. The crystal size in the obtained crystallization slurry was about 100 μm. In addition, 5-methyluridine and other impurity nucleic acid crystals were well separated by spontaneous precipitation and separated into two layers.

Example 4 When the upper layer of Examples 1 to 3 was separated from the lower layer of 5-methyluridine as a supernatant and the lower layer was centrifuged, all were separated at a high speed.

Example 5 Micrococcus luteus FERM P-7399 according to Example 2 of JP-A-2-23882.
After culturing, the bacterial cell culture solution was prepared, and then washed bacterial cells were obtained by a conventional method. Add Tris buffer to this to bring it back to its original volume,
Substrate 5-methyluracil and guanosine were added and the enzymatic reaction was carried out in the same manner as in Example 1 of the present invention. The resulting enzyme reaction solution was cooled, filtered, decolorized with activated carbon, filtered, and concentrated in vacuo to give 2-methyluridine (25.5%) and thymine (1.95).
% And guanosine 0.57% to obtain 384.9 g of a concentrated solution. While stirring this at 50 rpm, gradually add 10
It was cooled to 0 ° C. to precipitate crystals. This is left to stand for 10 minutes, the upper portion of the microcrystal suspension other than the lower 5-methyluridine layer is decanted and allowed to flow out, and the crystals in the effluent are separated by filtration to give 5-methyluridine per dry weight. 22.6%, thymine 50.7%, guanosine 1
8.62 g of wet crystals containing 0.7% were obtained. The mother liquor side was returned to the original container in which the 5-methyluridine crystal layer remained, stirred, and cooled again to 10 ° C. This was again decanted in the same manner as above, the crystals in the effluent were again filtered and separated, and 4-methyluridine was added to 4 times the dry weight.
1.4%, thymine 26.1%, guanosine 14.4
%, Guanine 0.052% in dry condition 4.57
g crystals were obtained. The mother liquor side was returned to the original container in which the 5-methyluridine crystal layer remained, cooled to 10 ° C. with stirring, and the whole fluid was separated by a basket type centrifugal filter. At the time of separation, the crystal layer was washed with 57.8 g of cold water. The separated crystals were dried to obtain 75.21 g of hydrated crystals. The yield of 5-methyluridine from the concentrate was 71.
4%. The purity of this crystal is 93.24%,
As impurities, 3.33% thymine and 0.342% guanosine were contained. The ratio of thymine to 5-methyluridine was 7.6% in the concentrated solution and 3.6% in the product, and was half in the crystallization and decantation steps. Furthermore, qualitatively, pseudoguanosine was also significantly reduced. Furthermore, by repeating the decantation, the impurity nucleic acid could have been further reduced. The average crystal grain size of 5-methyluridine in this crystallization was about 350 μm, and the average grain size of the microcrystals was 10 μm.

Example 6 After preparing a cell culture solution in the same manner as in Example 5 of the present invention,
An enzymatic reaction was performed. The enzyme reaction solution is cooled, filtered, decolorized with activated carbon, filtered, and concentrated in vacuo to give 5-methyluridine 2
580 L of a concentrated solution containing 3.0%, 1.71% thymine and 0.45% guanosine was obtained. This is 100 rp in a crystallizer
While stirring at m, the mixture was gradually cooled to 5 ° C. to precipitate crystals. After stirring was stopped and the 5-methyluridine crystals were allowed to settle for about 15 minutes, the upper crystallite suspension containing the crystallites was mechanically separated using a pump. The fine crystals in this separated liquid were separated by filtration, the mother liquor side was returned to the original crystallization container, stirred, and cooled again to 5 ° C. After repeating the above-mentioned agitation, standing, upper crystallite suspension separation, microcrystal filtration separation and resuspension, four times, the whole suspension was separated by a basket type centrifugal filter. The crystals were washed with 60 L of water during separation. The separated crystals were dried to obtain 96 kg of hydrated crystals. The yield of 5-methyluridine from the concentrate is 72.0.
%Met. The purity of this crystal was 93.8%, and 1.8% thymine and 0.16% guanosine were contained as impurities. The ratio of thymine to 5-methyluridine was 7.4% in the concentrated solution and 1.9% in the product, and was reduced to 1/4 in the crystallization and decantation steps.
Sudguanosine was also significantly reduced. Furthermore, by repeating the decantation, the impurity nucleic acid could have been further reduced. In addition, in this crystallization
The crystal average particle size of methyluridine was about 550 μm, and the average particle size of the microcrystals was 15 μm.

Comparative Example 1 Flavobacterium was prepared in the same manner as in Example 1 of the present invention.
A cell culture solution of Flavobacterium rhenanum FERM BP-1862 was prepared. Without removing the culture medium components of this cell culture medium, the substrates 5-methyluracil and guanosine were added, and the reaction was carried out at the temperature of 60 ° C. as in Example 1 of the present invention. The reaction solution was cooled, filtered, decolorized with activated carbon, filtered and concentrated in vacuo, and this was cooled to 5 ° C. Obtained 5-
The crystal size of methyluridine was 20 to 30 μm, and no separation phenomenon due to spontaneous precipitation with other impurity nucleic acid crystals was observed. Also, centrifugation took a very long time.

Comparative Example 2 In the same manner as in Example 5 of the present invention, an enzyme reaction was carried out after preparing a cell culture solution of Micrococcus luteus FERM P-7399. The obtained enzyme reaction solution was cooled, filtered, decolorized with activated carbon, filtered, and concentrated under vacuum to obtain 7.05 kg of a concentrated solution containing 25.2% of 5-methyluridine, 1.55% of thymine, and 0.42% of guanosine. . While further stirring, this was gradually cooled to 5 ° C. to precipitate crystals. Immediately after the suspension was allowed to stand, the whole amount was immediately separated by a basket type centrifugal filter to obtain 5-methyluridine.
5223 g of mother liquor containing 5%, thymine 0.26% and guanosine 0.12% was obtained. Further, the crystal layer was washed with 814 g of cold water. The mother liquor at the time of this washing contained 5.18% of 5-methyluridine, 0.31% of thymine, and 0.19% of guanosine, and was 1926 g. The separated crystals were dried to obtain 1.52 kg of hydrate crystals. The yield of 5-methyluridine from the concentrated liquid was 76.3%. The purity of this crystal was 89.28%, and the impurity was 5.
It contained 21% thymine and 0.69% guanosine. The ratio of thymine to 5-methyluridine was 6.2% in the concentrated solution and 5.8% in the product, and there was no reduction in the crystallization process.

[0048]

Industrially useful 5-methyluridine can be highly purified by a simple method without using a special solvent such as an organic solvent.

Claims (4)

[Claims] 1. A microorganism capable of producing 5-methyluridine from ribose-1-phosphate or a salt thereof and 5-methyluracil or a nucleoside, infinite phosphoric acid or a salt thereof and 5-methyluridine from 5-methyluracil. In a method for producing 5-methyluridine by an enzymatic reaction using a microorganism having the ability to: after culturing the microorganism, (a) removing some or all of the medium components, and then (b) the After allowing ribose-1-phosphate or its salt and 5-methyluracil or nucleoside, infinite phosphoric acid or its salt and 5-methyluracil to act on the microorganism to form 5-methyluridine, then (c) A method for producing 5-methyluridine, which comprises crystallizing and separating methyluridine 2. The method according to claim 1, wherein the removal rate of the medium components is 50% to 99%. 3. The crystal of 5-methyluridine is separated from other impurity crystals by a sedimentation speed difference when crystallizing and separating 5-methyluridine. The method described. 4. The method according to claim 1, wherein the separation method is a decantation method or a mechanical separation method.
The method according to claim 1.