JP3776160B2 - Method for producing D-calcium pantothenate - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a process for producing calcium D-pantothenate from a D-pantothenic acid fermentation broth produced directly by a microorganism. Calcium D-pantothenate is a useful vitamin widely used in the pharmaceutical, food and feed fields.
[0002]
[Prior art]
Conventionally, the manufacturing method of calcium pantothenate is roughly divided into a chemical synthesis method and a direct fermentation method. As a chemical synthesis method, D, L-pantolactone synthesized using isobutyraldehyde as a starting material is optically resolved by a chemical or enzymatic method, and the obtained D-pantolactone is condensed with β-alanine calcium to form D. -The process of obtaining calcium pantothenate is widely practiced on an industrial scale. As a direct fermentation method, a new production method in which D-pantothenic acid is directly produced by a microorganism using sugar and β-alanine as a raw material has recently been reported (Japanese Patent Laid-Open No. Hei 6-261773). Among them, as a method for producing calcium D-pantothenate from D-pantothenic acid direct fermentation broth, it was neutralized after desalting with an ion exchange resin and concentrated as a calcium salt, and after addition of methyl alcohol (methyl alcohol concentration 83v / V%) D-calcium pantothenate crystals are precipitated.
[0003]
[Problems to be solved by the invention]
The direct fermentation method is more efficient than the chemical synthesis method because it does not require optical resolution, but the fermented liquid is insoluble matter such as D-pantothenic acid and other monosaccharides, oligosaccharides, organic acids, proteins Since it contains various soluble impurities such as inorganic salts (cations, anions), the biggest problem is how to separate and purify high-purity calcium D-pantothenate from this fermentation broth with high yield. Met. In the method described in the published patent publication, after desalting with an ion exchange resin, D-pantothenic acid is neutralized and concentrated as a calcium salt, methyl alcohol is added (methyl alcohol concentration 83%), and D-pantothenic acid is added. A production method for precipitating calcium crystals has been described. (1) Monosaccharides and oligosaccharides contained in the fermentation broth cannot be separated and removed by ion-exchange resin treatment, and there are about 10% per pantothenic acid at the stage of the crystallization raw solution. Therefore, it causes color development due to heat at the time of concentration of the ion exchange resin treatment solution, and causes a decrease in the crystallization yield at the time of crystallization. Furthermore, in order to obtain a high crystallization yield, (2) Before adding methyl alcohol, increase the concentration of aqueous eluate so that the concentration of calcium pantothenate is 7 w / v% or higher and the methyl alcohol concentration is about 90 v / v% (D-calcium pantothenate about 50 w / v). ) It is necessary to concentrate, the viscosity of the D- calcium pantothenate solution in the vicinity of this concentration has drawbacks such fairly high concentration operation is difficult.
[0004]
[Means for Solving the Problems]
The inventors of the present invention have earnestly devised a method for producing high-quality calcium D-pantothenate efficiently and in a high yield from a culture solution obtained by a direct fermentation method and containing impurities such as sugar in addition to D-pantothenic acid. As a result of research, the present invention has been completed.
That is, the present invention is such that a solution containing D-pantothenic acid directly produced by fermentation by microorganisms is brought into contact with activated carbon to adsorb D-pantothenic acid on the activated carbon, and then eluted with a hydrophilic organic solvent, and then an alkali containing calcium. A method for producing calcium D-pantothenate, which comprises collecting calcium D-pantothenate that is neutralized with an agent and precipitated.
The solution containing D-pantothene produced directly by the microorganism in the present invention is a microorganism having the ability to synthesize D-pantothenic acid as described in JP-A-6-261773, for example, the intestinal bacterium Escherichia coli 814 / pFV31 ( IFO 15374, FERM BP4401) can be obtained, for example, by culturing in a medium using glucose as a sugar raw material, and contacting with β-alanine. At this time, the production amount of D-pantothenic acid is usually 40 g / liter. The solution is usually removed by removing insoluble solids such as bacterial cells by a method such as centrifugation or filtration to remove insoluble solids in the liquid, thereby preventing charcoal contamination in the subsequent activated carbon treatment, extending the life of the charcoal, etc. From the point of view is desirable.
The solution containing D-pantothenic acid from which insoluble solids such as bacterial cells have been removed is adjusted to a pH of usually 1 to 5, preferably pH 2 to 4, with an inorganic acid such as hydrochloric acid or sulfuric acid. When the pH is less than 1, the decomposition of D-pantothenic acid is likely to occur and the yield is lowered. When the pH is more than 5, the amount of D-pantothenic acid adsorbed on the activated carbon decreases, while the sugar adsorption power increases. The separation and removal of saccharides is worsened.
[0005]
The activated carbon used in the present invention is not particularly limited, and any commercially available carbon can be used as long as it is usually marketed for liquid phase separation. For example, the total pore volume of pores having a diameter of 300 mm or less (hereinafter simply referred to as “pore volume”) is 0.4 cc / g or more and the average pore diameter of pores having a diameter of 300 mm or less (hereinafter simply referred to as “average pore diameter”). And activated carbon having a pore characteristic of 17 mm or more. The activated carbon having such specific pore characteristics is, for example, 1) after immersing wood raw materials such as wood pieces and coconut husks in chemicals such as zinc chloride, phosphoric acid, calcium chloride and baking at about 600 to 700 ° C. For example, by desorbing and washing additive chemicals with an acid such as hydrochloric acid, or 2) treating mineral raw materials such as coal and petroleum pitch with alkali, and then using 750-900 with steam, carbon dioxide gas, etc. It is obtained by activating at ° C. The activated carbon may be in the form of powder, granule or granule, but when packed in a column, it is preferably granule or granule in order to suppress tower pressure. Specific examples of the activated carbon include granular white KLH (manufactured by Takeda Pharmaceutical, pore volume 1.09cc / g, average pore diameter 32mm), granular white powder W (manufactured by Takeda Pharmaceutical, pore volume 0.49cc / g, average pore diameter 18mm) , Granular white LH2C (manufactured by Takeda Pharmaceutical, pore volume 0.74cc / g, average pore diameter 19mm), CAL (produced by Calgon Corporation, pore volume 0.55cc / g, average pore diameter 21mm), granular activated carbon Diahop 008 (Mitsubishi Chemical) Manufactured, pore volume 0.61 cc / g, average pore diameter 20 mm).
As a method of bringing the liquid to be treated into contact with the activated carbon, for example, a fixed bed adsorption method, that is, a method of flowing the liquid to be treated through the activated carbon packed in the column is suitable. According to this method, impurities can be separated and removed in a chromatographic manner, and fractionation of the eluate of D-pantothenic acid with a hydrophilic organic solvent can be facilitated. Practically, so-called series adsorption is preferred in which the liquid to be treated is passed through two or more activated carbon packed columns in series to adsorb D-pantothenic acid to the activated carbon.
[0006]
The adsorption treatment method will be described taking the case of using two columns as an example, that is, as shown in FIG. 1, the activated carbon packed column A column and B column are connected in series and the liquid passage (i) → (ii) → (iv) The liquid to be treated is flowed in the order of (v) → (vi) and passed until the pantothenic acid concentration at the outlet of the A tower becomes the same as the concentration at the inlet of the A tower. If the concentrations at the outlet and the inlet are the same, the tower A and the tower B are separated, the liquid flow is switched to the tower B, another activated carbon tower is connected to the tower B in series, and the pantothenic acid concentrations at the outlet and the inlet of the tower B are the same. Pass until it becomes.
Fig. 2 shows D-pantothenic acid when D-pantothenic acid direct fermentation broth is decolorized with activated carbon for cell separation and decolorization and passed through a solution containing D-pantothenic acid adjusted to pH 3 with hydrochloric acid. And the component leakage curve of saccharides. The point where the concentration of pantothenic acid at the outlet of the tower A is the same as that at the inlet is the saturated adsorption point (b), which is about 1.5 when compared with the breakthrough adsorption point shown by (a) where there is no leakage of D-pantothenic acid. Double D-pantothenic acid is adsorbed (about 200 g / liter-LH2C). D-pantothenic acid that has started to leak after the breakthrough adsorption point is adsorbed in the next column. At this time, the adsorption power of saccharides and other impurities coexisting in the liquid to be treated to activated carbon is weaker than that of D-pantothenic acid, so it is expelled by D-pantothenic acid during the passage and the leakage rate reaches 250% and then reaches the saturated adsorption point. It has been found that 90% or more of the sugars in the liquid to be treated are separated and removed by the treatment. This was a completely unexpected new finding for those skilled in the art. In addition, inorganic salts (cations and anions) contained in the liquid to be treated can be separated and removed by flowing into the adsorption waste liquid without being adsorbed on the activated carbon.
[0007]
Next, a hydrophilic organic solvent is passed through the activated carbon column on which D-pantothenic acid is saturated and adsorbed to elute D-pantothenic acid. As the hydrophilic organic solvent, a lower alcohol having 1 to 5 carbon atoms such as methyl alcohol, ethyl alcohol, isopropyl alcohol and the like can be advantageously used, but methyl alcohol is particularly advantageous for crystallization in a subsequent step. That is, the crystals of calcium D-pantothenate are in a polymorphic form, depending on the solvent environment, α, β, γ crystals, 4 molecules of MeOH · 1 molecule of H ₂ O (4MeOH · 1H ₂ O crystal), and amorphous form. However, the 4MeOH · 1H ₂ O crystals precipitated from the crystallization stock solution when methyl alcohol is used as the elution solvent are granular and have very good separability. The elution temperature is 10 to 30 ° C, preferably 20 to 30 ° C.
The situation in which D-pantothenic acid is eluted from the activated carbon column using methyl alcohol will be described with reference to FIG. 3 showing the component elution curve in Example 1.
The first 0.7 vol elution section (vs. LH2C charcoal) was the section where the water retained in the column was pushed out, and the D-pantothenic acid concentration in this section was only 0.4% w / v%. In the next 1.5 vol elution section, the D-pantothenic acid concentration was 8.7 w / v% (migration amount 90%), and the water content was 7.5 w / v%. Since the D-pantothenic acid concentration of the liquid to be treated was 2.6 w / v%, this elution section was concentrated about 3.4 times by adsorption and elution. Furthermore, the last 0.8 vol elution section had a D-pantothenic acid concentration of 0.9 w / v% (migration amount 4%) and a water content of 0.05 w / v%.
[0008]
The hydrophilic organic solvent concentration and the D-pantothenic acid concentration in the eluate are usually 80 to 98 v / v%, preferably 85 to 95%, more preferably 85 to 95% in relation to the crystallization efficiency in the subsequent crystallization operation. Preferably, it is 90 to 92%, and the D-pantothenic acid concentration is preferably 7 w / v% or more. Therefore, the 1.5 vol elution section can be neutralized as it is to obtain a crystallization stock solution.
Next, the eluate is neutralized with an alkaline agent containing calcium. Any alkaline agent may be used as long as it contains sufficient calcium to neutralize D-pantothenic acid contained in the eluate, but calcium hydroxide is preferred. More practically, approximately equimolar powdered calcium hydroxide is added to the eluate with respect to D-pantothenic acid. When unreacted calcium hydroxide fine powder appears, it is better to remove it by filtration. Neutralization should keep the liquid temperature at 15 ° C. or higher in order to prevent crystal precipitation of calcium D-pantothenate.
[0009]
The crystallization undiluted solution thus prepared is preferably cooled to 10 ° C. or lower, desirably 5 ° C. or lower, and about 0.2% of seed crystals are added to calcium D-pantothenate and stirred at 0 to 5 ° C. If left for 10 hours or longer, calcium pantothenate crystals can be obtained in good yield. The crystallization slurry is collected by centrifugal separation using a conventional centrifugal dehydrator or the like, or filtration using a filter press to obtain wet crystals.
When methyl alcohol is used as the elution solvent, the wet crystals contain about 25 w / w% methyl alcohol and about 5 w / w% moisture. The wet crystals can be dried to a moisture content of about 0.5% when dried under reduced pressure at 70 to 80 ° C. If the moisture crystals are further dried using humidity-controlled air (80 ° C., RH 20%), the moisture content is about 2%. % D-calcium pantothenate powder substantially free of methyl alcohol can be obtained. Alternatively, a solution obtained by dissolving wet crystals in water and concentrating / desolving to a concentration of about 50 w / v% can be spray-dried by a conventional spray dryer to obtain calcium D-pantothenate powder. All crystal forms of the dried product are converted to amorphous forms.
As described above, this method allows saturated adsorption of D-pantothenic acid on activated carbon by passing a solution containing D-pantothenic acid through an activated carbon column, and at the same time, can effectively separate and remove inorganic ions and saccharides. Elution with a hydrophilic organic solvent and selecting a category taking into consideration the solvent concentration and D-pantothenic acid concentration can be used as a starting stock solution for the neutralization and crystallization process, and separation and collection after crystallization are easy. This is an extremely efficient method for producing high-purity calcium D-pantothenate.
[0010]
【Example】
The contents of the present invention will be described more specifically with reference to the following examples. However, these are only examples of the contents of the present invention and do not limit the scope of the present invention. Example 1
Escherichia coli IFO 814 / pFV31 strain was cultured in a conventional manner in a 5-liter jar fermenter using a medium containing glucose as a carbon source to obtain 2.5 liters of a D-pantothenic acid direct fermentation broth. Filtration was performed at 40 ° C. using a ceramic filter (manufactured by Toshiba Ceramics Co., Ltd.) having a pore size of 0.1 μm to obtain 1.67 liters of filtrate from which insoluble solids such as bacterial cells were removed. This liquid contained 38.5 mg / ml (64.3 g) of D-pantothenic acid and 10.3 mg / ml of sugar (total sugar by phenol sulfuric acid method) (about 27% per D-pantothenic acid). The filtrate was passed through a column (packing volume 500 ml) with an inner diameter of 70 mm and a height of 130 mm packed with activated carbon K-1 for decolorization (manufactured by Takeda Pharmaceutical, pore volume 1.12 cc / g, average pore diameter 32 mm) and washed with water. In addition, 2.4 liters of a liquid having a decolorization rate of 99.5% was obtained. Concentrated hydrochloric acid (56 ml) was added to this solution to adjust the pH to 3.0, and a column (packing volume) with an inner diameter of 50 mm and a height of 100 mm packed with activated carbon for liquid phase separation and LH2C charcoal (granular, steam activated charcoal, manufactured by Takeda Pharmaceutical). 200 ml) 2 towers (A and B towers) were connected in series and passed continuously. The amount of liquid passing through the tower A up to the saturation adsorption point was 2.2 liters. By this operation, 29 g (145 g / liter-LH2C charcoal) of D-pantothenic acid was adsorbed on the A column. The tower A and the tower B are separated after the saturation adsorption point, and the remaining liquid is passed through the tower B. Tower A was washed with 600 ml of water, and D-pantothenic acid was eluted with methyl alcohol. That is, the first 0.7 vol · 140 ml was discarded (D-pantothenic acid loss was 2%), and the next 1.5 vol · 300 ml (D-pantothenic acid transfer amount was about 90%) was advanced to the crystallization step. The final 0.8 vol · 160 ml (4% transfer amount of D-pantothenic acid) was used as an eluent for the next column to recover D-pantothenic acid.
D-pantothenic acid content of the above 1.5 vol. 300 ml section is 8.7 w / v% (26.3 g), total sugar content is 0.43 w / v% (5% per D-pantothenic acid), and methyl alcohol concentration Was 92.5 v / v%. 4.8 g of calcium hydroxide powder was added to this elution section and stirred sufficiently to neutralize D-pantothenic acid to obtain a calcium salt (28.6 g of calcium D-pantothenate). Next, it was filtered through a Nutsche pre-coated with diatomaceous earth to obtain a crystallization stock solution. These operations were performed at 20 to 25 ° C. Transfer the crystallized stock solution to a round bottom flask equipped with a stirrer, cool to 5 ° C, add 57 mg of seed crystals (0.2% calcium D-pantothenate), cool to 2 ° C, hold for 15 hours and crystallize. Went. The crystallization slurry was filtered through a 3G glass filter, sprayed with methyl alcohol at 5 ° C., washed, 46.6 MeOH with one molecule of H ₂ O, 36.6 g of wet crystals with a MeOH content of 26% and a moisture content of 4%. (34.4 g of calcium D-pantothenate: crystallization yield 90%) was obtained. The wet crystals were dried at 80 ° C. under reduced pressure in the usual manner, and further air-conditioned at 80 ° C./RH 20% to obtain 26.1 g of a D-calcium pantothenate dry powder having a moisture content of 2.4%. This product complies with Japanese, US and UK standards for solution (clarity, color), calcium content, nitrogen content, specific rotation, crystal form, etc.
[0011]
【The invention's effect】
According to the present invention, a solution containing D-pantothenic acid directly produced by fermentation by a microorganism is brought into contact with activated carbon to efficiently adsorb D-pantothenic acid, thereby efficiently removing impurities, particularly sugars, and then methyl alcohol and the like. By eluting with a hydrophilic organic solvent and performing the crystallization operation as it is, high-quality calcium D-pantothenate can be obtained from the fermentation filtrate with high efficiency and high yield.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of a fixed bed in which activated carbon packed columns A column and B column are connected in series, and (i) to (vi) indicate liquid flow paths.
FIG. 2 is a component leakage curve when a liquid to be treated is passed through an LH2C charcoal column. A thin solid line A shows a breakthrough curve for D-pantothenic acid, and a thick solid line B shows a breakthrough curve for sugar. (a) shows the breakthrough adsorption point of D-pantothenic acid, and (b) shows the saturated adsorption point of D-pantothenic acid.
FIG. 3 shows a component elution curve from an LH2C charcoal column using methyl alcohol as an elution solvent. The thin solid line A is D-pantothenic acid, the thick solid line B is moisture, and the dotted line C is a sugar elution curve.

Claims (5)

Separating insolubles from the fermentation broth of D-pantothenic acid directly fermented by microorganisms;
Decolorizing the fermentation broth using activated carbon to obtain a solution containing D-pantothenic acid;
Adjusting the pH of the solution containing D-pantothenic acid to 1-5;
A solution containing D-pantothenic acid is continuously brought into contact with granular or granular activated carbon packed in a column in which two or more saccharides have lower adsorption power than D-pantothenic acid and are connected in series. Adsorb pantothenic acid on activated carbon to the saturation adsorption point;
Eluting D-pantothenic acid with a hydrophilic organic solvent;
Neutralize the eluate with an alkaline agent containing calcium to precipitate calcium D-pantothenate;
Collecting calcium D-pantothenate;
The manufacturing method of the calcium D-pantothenate characterized by including a process.   The process according to claim 1, wherein the hydrophilic organic solvent is a lower alcohol having 1 to 5 carbon atoms.   The process according to claim 2, wherein the lower alcohol is methyl alcohol.   The process according to claim 1, wherein the alkaline agent containing calcium is calcium hydroxide.   The hydrophilic organic solvent is methyl alcohol, the alkaline agent containing calcium is calcium hydroxide, and the calcium D-pantothenate is collected as a crystal having 4 molecules of methyl alcohol and 1 molecule of water. Manufacturing method.

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