JP2877366B2 - Method for producing crystalline L-ascorbic acid-2-phosphate sodium salt - Google Patents

Description

The present invention relates to L-ascorbic acid-2-phosphate (hereinafter referred to as L-ascorbic acid-2-phosphate).
Abbreviated as AsA2P. ) Crystals (hereinafter abbreviated as Na salts) and a method for producing the same.

Crystals of AsA2PNa salt are useful as stabilized derivatives of ascorbic acid and can be used in pharmaceuticals, foods, cosmetics, and various other industrial fields.

2. Description of the Related Art Conventionally, many reports on AsA2P have been known. For example, vitamins (Japan) Volume 41 (1970)
Pages 387-398 describe the chemistry and application of ascorbic acid phosphates, and pages 390 specifically describe the properties of magnesium, sodium and calcium salts of ascorbic acid-3-phosphate. . The property of sodium ascorbic acid-3-phosphate is described as a colorless powder which is difficult to crystallize. In addition, ascorbic acid-3-phosphate in this document has been corrected to AsA2P on page 185 of Vitamin 51 (1985).

Other methods for purifying AsA2P include a method using activated carbon (JP-A-59-51293 and JP-A-59-106494), a method using a strongly acidic cation exchange resin, and a method using a strongly basic anion exchange resin (see No. 45-4497), a method using a weakly basic or medium basic anion exchange resin (JP-A-62-30791),
Method using a weak acid type chelate resin (Japanese Patent Laid-Open No. 62-103096)
Such methods are known.

JP-A-59-51293 discloses that purified As
It is stated that the A2P is preferably in the form of a salt after decationization. As the salt form, magnesium salt, calcium salt, sodium salt, potassium salt and the like are described. Furthermore, it is described that the liquid containing the target substance obtained in this manner is subjected to a concentration step and a crystallization step which are usually performed, but all the examples are directed to magnesium salts, and to the sodium salt crystals. Is not written. In addition, the present inventor tried to obtain sodium salt crystals by the method described in this publication, but could not produce crystals but merely produced candy.

Problems to be Solved by the Invention AsA2P is generally put into practical use in the form of an Mg salt, but the following points are problematic in the purification process and in the use of the product.

Since AsA2PMg salt is amorphous, it is difficult to obtain high purity AsA2PMg salt by crystallization. In particular, the unreacted reaction raw material and the phosphorus compound generated as a by-product form an insoluble Mg salt, making purification difficult.

Since AsA2PMg salt is amorphous, it has a high water content and a high solvent residual ratio, and it is not easy to remove adhering water and solvent by drying.

AsA2PMg salt has a slow dissolution rate in water and tends to be candy-like. Therefore, to dissolve it in an industrially practical concentration,
It is necessary to devise a dispersion method at the time of dissolution.

When an AsA2PMg salt is used, in a system in which water is present, the color gradually increases over time.

Therefore, development of a stable and easy-to-handle AsA2P is desired.

Means for Solving the Problems According to the present invention, stable and easy to handle, high purity AsA2
P is provided as a crystal of the new AsA2PNa salt.

The crystals of the AsA2PNa salt of the present invention are prepared by adjusting the solution containing AsA2P to pH 8 to 10 with sodium hydroxide,
While heating and refluxing at a temperature of 0 to 80 ° C, an organic solvent selected from a lower aliphatic alcohol having 5 or less carbon atoms, an aliphatic saturated ketone having 5 or less carbon atoms and a cyclic ether has a concentration of 30 to 80% in a solution. (V / v), gradually added to the solution over 4 to 7 hours, and then left at a temperature of 30 ° C. or lower.

 The physical properties of the crystals of the AsA2PNa salt of the present invention are shown below.

(1) Elemental analysis value (2) molecular weight; 331.0 (C ₆ elemental analysis H ₆ O ₉ PNa ₃ · 1
/ 2H ₂ 0 and the estimated) (3) X-ray diffraction diagram; showing a Cu-K X-ray diffraction pattern obtained by measuring in a powder X-ray diffraction method using a _two-wire in Figure 1. The numbers in the figure correspond to the numbers in Table 1. The comparative intensity is represented with the peak 2 in the figure as 100.

(4) Infrared absorption spectrum; FIG. 2 shows an infrared absorption spectrum measured by the KBr tablet method.

(5) Solubility in solvents; easy to dissolve in water, but poorly soluble in lower alcohols and ketones. Hardly soluble in ether.

 FIG. 3 shows the comparative dissolution rates of Na salt and Mg salt.

(6) distinction of basic, acidic and neutral; weakly basic (7) color of substance; colorless and transparent (8) crystal form; usually columnar.

As the starting solution used in the present invention, any solution such as an AsA2P solution, a metal salt solution of AsA2P, or an alkaline earth metal salt solution can be used as long as the solution contains AsA2P. For example, an AsA2P-containing liquid produced by the action of an enzyme or a microorganism from ascorbic acid and a phosphate donor can be suitably used. Metal salts include potassium salts, and alkaline earth metal salts include magnesium salts and calcium salts.

When AsA2P is in the form of a salt, or when the AsA2P-containing solution contains a metal ion such as an alkali metal or an alkaline earth metal, it is desirable to treat the aqueous solution with an appropriate ion exchange resin to decationize. After AsA2P is adsorbed on an ion exchange resin and eluted with 0.1 to 2N dilute hydrochloric acid, the pH is adjusted with sodium hydroxide.

The pH adjustment with sodium hydroxide is usually performed with a 5 to 15N aqueous sodium hydroxide solution. In the solution after pH adjustment
AsA2P concentration is 30-100 g / l, preferably 50-80 g / l,
If necessary, it is diluted with water or concentrated by vacuum concentration or the like.

Then, the solution is heated to reflux at a temperature of 40 to 80 ° C., preferably 50 to 70 ° C., and the organic solvent is concentrated to 30 to 80% (v / v).
The crystals are crystallized by slow addition over 4 to 7 hours as in v). If necessary, further heat reflux to 2-10
Continue for hours.

Examples of the organic solvent to be used include lower aliphatic alcohols having 5 or less carbon atoms such as methanol and ethanol, saturated aliphatic ketones having 5 or less carbon atoms such as acetone, and cyclic ethers such as tetotehydrofuran. These organic solvents may be used alone or in combination.

When left at a temperature of 30 ° C. or less after heating and refluxing, the crystallized crystals grow and mature. The crystals are isolated by filtration, washed with the above-mentioned organic solvent, and then subjected to treatment such as vacuum drying to obtain high-purity white crystalline AsA2PNa salt.

According to the method of the present invention, the unreacted phosphorus compound in the reaction solution of the AsA2P formation reaction forms a salt having high solubility with sodium, so that the amount of the unreacted phosphorus compound mixed into the product is small, the separability of crystals from the crystallization mother liquor is good, and The number of crystallization operations is small.

The obtained crystals have a low water content, easy removal of adhering water and solvent, and a high dissolution rate.

 The embodiments of the present invention will be described with reference to examples.

Example 1 Pseudomonas azotocolligans ATCC 12417 was prepared using polypeptone 10 g / l, meat extract 7 g / l, yeast extract 5 g / l and N
30 ml of a medium (hereinafter abbreviated as KM102 medium) adjusted to pH 7.2 containing 3 g / l of aCl was inoculated and cultured at 30 ° C. for 20 hours. Then, 12 ml of the seed culture obtained above was inoculated into 300 ml of KM102 medium, and cultured at 30 ° C for 20 hours. The resulting culture was
Centrifugation at 00 × g for 10 minutes to obtain 11.8 g of wet cells,
Stored frozen at 0 ° C. 200 mM ascorbic acid, potassium pyrophosphate 2 containing 50 mg / ml of cryopreserved cells (wet cell weight)
100 mM of a 100 mM sodium acetate buffer (pH 4.0), 4 g / l of Nimeen S-215 (manufactured by NOF CORPORATION), and 50 ml of a reaction solution having a composition of 10 ml / l of xylene were subjected to 100 r with a magnetic starter.
While stirring at pm, adjust the pH to around 4.0 with sodium hydroxide,
The reaction was performed for 36 hours while maintaining the temperature at 40 ° C. The obtained reaction solution (containing 30 g / l of AsA2P.1) was gradually sterilized, and the solution was passed through a column filled with Prolite A100 (a neutral anion exchange resin, manufactured by Prolite International) 1. The adsorbed AsA2P was used. Was eluted with 1N diluted hydrochloric acid.

The solution obtained by the elution was adjusted to pH 9.5 with a 10N aqueous sodium hydroxide solution.

The solution was concentrated under reduced pressure to a concentration of AsA2P of 80 g / l and passed through a 0.45 micron Millipore filter. Liquid at 60 ℃
While heating to reflux and stirring, 900 ml of methanol was added slowly over about 6 hours, and then heating and refluxing were continued for 3 hours. It was left overnight at 30 ° C or lower. The resulting crystals were collected and washed with methanol. Then, vacuum-dry at 40 ° C overnight to purify the desired crystal of AsA2PNa salt 21.4 g (yield 71%)
I got Table 2 shows the product analysis values of the purified product obtained.

Example 2 AsA2P 20.0 g / l obtained by the same method as in Example 1
The reaction solution containing
, And 4 g of powdered activated carbon was added. The mixture was allowed to stand at 50 ° C. for 30 minutes and decolorized. The solution was adjusted again to pH 9.5 with a 1N aqueous sodium hydroxide solution, and concentrated under reduced pressure to 80 g / L AsA2P. 600 ml of acetone was added slowly over 6 hours while heating and refluxing 300 ml of the concentrated solution at 60 ° C., refluxed for 3 hours, and then left at 5 ° C. for 3 hours. The resulting crystals were collected and washed with acetone. Then, vacuum drying was carried out at 40 ° C. overnight, and 16.5 g of a purified AsA2PNa crystal (yield 80
%).

Example 3 50.0 g of AsA2PMg salt (unrefined product) was dissolved in purified water 1. This solution was passed through a column filled with 500 ml of Diaion SK-1B (strongly acidic cation exchange resin, manufactured by Mitsubishi Kasei),
Decationized. Effluent with 1N aqueous sodium hydroxide
The mixture was adjusted to pH 9.5, decolorized with 5 g of powdered activated carbon, and passed through. The liquid was crystallized and dried in the same manner as in Example 1 to obtain 42.5 g (yield: 85%) of a purified AsA2PNa salt crystal. Table 2 shows the product analysis values of the purified product obtained.

Comparative Example 20.0 g of AsA2P obtained by the same method as in Example 1
A 1N diluted hydrochloric acid solution containing / l was adjusted to pH 8 with a 1N magnesium hydroxide solution. Decolorize with powdered activated carbon and remove AsA
The solution was concentrated under reduced pressure until the concentration of 2P became 50 g / l, and 900 ml of methanol was slowly added thereto over 6 hours while heating and stirring the concentrated solution at 60 ° C. Reflux for an additional 3 hours and cool for 30 nights
Leave the precipitate at room temperature below ℃, dry and dry AsA2PM
22.5 g (75% yield) of purified salt product was obtained. Table 2 shows the product analysis values of the purified product obtained.

Effects of the Invention According to the present invention, crystals of an AsA2PNa salt are provided with high purity and high yield.

[Brief description of the drawings]

FIG. 1 is a powder X-ray diffraction pattern of the AsA2PNa salt crystal of the present invention. FIG. 2 is an infrared absorption spectrum of the AsA2PNa salt crystal, and FIG. 3 is a comparison of the dissolution rates of the AsA2PNa salt crystal and the AsA2PMg salt. In the figure, 1 is AsA2PNa salt crystal, 2 is A
2 shows sA2PMg salt. The vertical axis in FIG. 1 shows the diffraction intensity and the horizontal axis shows the diffraction angle, and the vertical axis in FIG. 2 shows the transmittance and the horizontal axis shows the wave number. The vertical axis in FIG. 3 shows the AsA2P concentration in the supernatant, and the horizontal axis shows the stirring and dissolving time. FIG. 4 is a photograph (× 200) of an AsA2PNa salt crystal.

Claims (6)

(57) [Claims] 1. A solution containing L-ascorbic acid-2-phosphate is adjusted to pH 8 to 10 with sodium hydroxide, and the solution is heated and refluxed at a temperature of 40 to 80.degree.
An organic solvent selected from the following lower aliphatic alcohols, aliphatic saturated ketones having 5 or less carbon atoms, and cyclic ethers is added over 4 to 7 hours so that the concentration in the solution becomes 30 to 80% (v / v). A crystalline L-ascorbic acid-2-sodium phosphate characterized by crystallizing L-ascorbic acid-2-phosphate sodium salt by being gradually added to the solution and then allowed to stand at a temperature of 30 ° C. or lower. Method for producing salt. 2. A solution containing a metal salt of L-ascorbic acid-2-phosphate or an alkaline earth metal salt is decationized, and then adjusted to pH 8 to 10 with sodium hydroxide.
While heating and refluxing the solution at a temperature of 40 to 80 ° C, an organic solvent selected from a lower aliphatic alcohol having 5 or less carbon atoms, an aliphatic saturated ketone having 5 or less carbon atoms and a cyclic ether having a concentration of 30 to The solution was gradually added to the solution over 4 to 7 hours so as to become 80% (v / v), and then left at a temperature of 30 ° C. or less to crystallize L-ascorbic acid-2-phosphate sodium salt. A method for producing crystalline L-ascorbic acid-2-phosphate sodium salt, characterized in that: 3. The method for producing crystalline sodium L-ascorbic acid-2-phosphate according to claim 2, wherein the metal salt is a potassium salt. 4. The crystalline L- according to claim 2, wherein the alkaline earth metal salt is a magnesium salt or a calcium salt.
A method for producing ascorbic acid-2-phosphate sodium salt. 5. The crystalline L-ascorbic acid-2-phosphate according to claim 1, wherein heating and refluxing are further continued for 2 to 10 hours after adding the organic solvent. Method for producing sodium salt. 6. The method for producing crystalline L-ascorbic acid-2-phosphate according to claim 1, wherein the organic solvent is at least one of methanol, ethanol, acetone and tetrahydrofuran.