METHOD FOR PRODUCING COENZYME Q10

Abstract

Methods for stably producing coenzyme Q10 are provided, which efficiently remove impurities derived from a microorganism in an extract of a coenzyme Q10-producing microorganism. A method for producing coenzyme Q10 includes: cooling a hydrophobic organic solvent extract of a coenzyme Q10-producing microorganism or a concentrated liquid of the hydrophobic organic solvent extract; and separating/removing the solid precipitated in the cooling step, wherein the hydrophobic organic solvent extract or the concentrated liquid has a water content of 50 ppm by weight or more and 1% by weight or less. Preferably, the extract obtained by extraction from a coenzyme Q10-producing microorganism in an organic solvent is brought into contact and mixed with an aqueous alkaline solution and then washed with water, where a resulting extract is concentrated to obtain the concentrated liquid, and the concentrated liquid is subjected to the cooling step.

Claims

1-7. (canceled)

8. A method for producing coenzyme Q10 comprising: (a) providing a first extract, wherein the first extract comprises (i) a hydrophobic organic solvent extract having a coenzyme Q10-producing microorganism or (ii) a concentrated liquid of an initial hydrophobic organic solvent extract, and wherein the hydrophobic organic solvent extract or the concentrated liquid has a water content of 50 ppm by weight or more and 1% by weight or less; (b) cooling the first extract under conditions effective to precipitate a solid; and (c) separating and removing the solid that was precipitated in (b).

9. The method according to claim 8, wherein the concentrated liquid of an initial hydrophobic organic solvent extract in (a) is obtained by: (a) mixing the initial hydrophobic organic solvent extract having the coenzyme Q10-producing microorganism with an aqueous alkaline solution, and then washing with water to provide a washed extract, (a) concentrating the washed extract, to obtain the concentrated liquid of the hydrophobic organic solvent extract.

10. The method according to claim 9 comprising: repeating a procedure in which, the solid that was separated and removed in (c), is added to a second extract having the coenzyme Q10-producing microorganisms, mixed with a second aqueous alkaline solution, washed with water, and then subject to a second cooling step; or repeating a procedure in which, a second extract having the coenzyme Q10-producing microorganisms is mixed with a second aqueous alkaline solution, the solid that was separated and removed in (c) is added to a second extract, a second extract is washed with water, and a second extract is subject to a second cooling step.

11. The method according to claim 8, wherein the cooling is conducted at a temperature of 20 C. or lower.

12. The method according to claim 8, wherein a concentration of coenzyme Q10 in the hydrophobic organic solvent extract or the concentrated liquid at the time of the cooling in the cooling step is 0.1 g/L or more and 300 g/L or less.

13. The method according to claim 8, wherein the hydrophobic organic solvent comprises a hydrocarbon solvent, a fatty acid ester solvent, or mixtures thereof.

14. The method according to claim 8, wherein the solid is separated and removed from the first extract by a rotary filter.

Description

EXAMPLES

[0094] Hereinafter, the present invention is described in more detail with Examples and Comparative Examples but is not restricted to the following Examples. In addition, the yield and purity of coenzyme Q10 in Examples and Comparative Examples do not represent the limiting value of the present invention nor the upper limit. The concentration of coenzyme Q10 was measured by high-performance liquid chromatography (HPLC) (manufactured by SHIMADZU) in the following condition.

HPLC Measurement Condition

Column: YMC-Pack ODS-A

[0095] Oven temperature: 30 C.
Mobile phase: methanol/hexane=85/15 by volume
Flow rate: 1.0 ml/min

Detection: UV 275 nm

[0096] A purity improvement percent point (% pt.) of coenzyme Q10 was calculated as the difference of weight percent of Q10 in two non-volatile constituents obtained by drying the solutions before and after the separation and removal of solid.

[0097] A removal rate of ergosterol was calculated by measuring ergosterol concentrations in the solutions before and after the separation and removal of solid and using the following formula. The ergosterol concentration is described as ERG concentration and was measured by using HPLC in the same condition as the above-described measurement condition of coenzyme Q10 concentration.

Removal rate of ergosterol (ERG)={(ERG concentration before the separation and removal of solidERG concentration after the separation and removal of solid)/(ERG concentration before the adsorption treatment)}100.

[0098] The water content was measured using Karl Fischer (AQUACOUNTER AQ-2100 manufactured by Hiranuma Sanygo Co., Ltd.).

Example 1

[0099] Saitoella complicata IFO10748 strain, which could produce coenzyme Q10, was aerobically cultivated in a culture medium (peptone 5 g/L, yeast extract 3 g/L, malt extract 3 g/L, glucose 20 g/L, pH 6.0) at 25 C. for 160 hours. The obtained microorganism culture medium containing coenzyme Q10 was concentrated by centrifugation, and the microorganism was homogenized by a pressure homogenizer. To the obtained microorganism homogenate were added 1.8 times amount of hexane and 0.7 times amount of 2-propanol to the volume of the microorganism homogenate, and the mixture was stirred at 40 C. for 1 hour to extract coenzyme Q10. To the extract of the coenzyme Q10-producing microorganism were added 8 vol % of a 4 wt % aqueous solution of sodium hydroxide relative to the extract, and 0.5 vol % of a 7% aqueous hydrogen peroxide relative to the extract as an oxidant. The mixture was stirred for 3 minutes and then allowed to stand to separate an aqueous layer. To the extract after the separation was added 13 vol % of tap water relative to the extract, and the resulting extract was stirred and washed with water. After repeating the washing twice, the extract was concentrated so that the coenzyme Q10 concentration was adjusted to 50 g/L. The reduced coenzyme Q10 ratio in the concentrate, i.e. the ratio of reduced coenzyme Q10 in the total coenzyme Q10, was 0 wt %. The concentrated liquid having a water content of 663.4 ppm by weight (the same applies hereinbelow) was cooled to 20 C. and subjected to suction filtration to separate and remove a solid. For the filtration, a Kiriyama funnel and No. 5-C filter paper for use in the Kiriyama funnel were used. As a result of analysis of the filtrate, it was confirmed that the water content in the filtrate was 201.8 ppm, the removal rate of ergosterol was 34.8%, and the purity of coenzyme Q10 was increased by 4.0% pt.

Example 2

[0100] The same concentrated liquid (water content: 663.4 ppm) of the coenzyme Q10-producing microorganism as that of Example 1 was cooled to 15 C. and subjected to suction filtration in the same manner as in Example 1 to separate and remove a solid. As a result of analysis of the filtrate, it was confirmed that the water content was 165.44 ppm, the removal rate of ergosterol was 39.0%, and the purity of coenzyme Q10 was increased by 4.9% pt.

Example 3

[0101] The same concentrated liquid (water content: 663.4 ppm) of the coenzyme Q10-producing microorganism as that of Example 1 was cooled to 10 C. and subjected to suction filtration in the same manner as in Example 1 to separate and remove a solid. As a result of analysis of the filtrate, it was confirmed that the water content was 107.2 ppm, the removal rate of ergosterol was 46.3%, and the purity of coenzyme Q10 was increased by 5.3% pt.

Example 4

[0102] The same concentrated liquid (water content: 663.4 ppm) of the coenzyme Q10-producing microorganism as that of Example 1 was cooled to 2 C. and subjected to suction filtration in the same manner as in Example 1 to separate and remove a solid. As a result of analysis of the filtrate, it was confirmed that the water content was 63.8 ppm, the removal rate of ergosterol was 51.0%, and the purity of coenzyme Q10 was increased by 5.8% pt.

Example 5

[0103] The concentrated liquid (water content: 585 ppm) of the coenzyme Q10-producing microorganism was prepared in the same manner as in Example 1, cooled to 10 C., and subjected to centrifugation at 9000 g for 5 minutes to recover a supernatant. Allegra X-22R CENTRIGUGE manufactured by Beckman Coulter, Inc. was used for the centrifugation. It was confirmed that the water content in the concentrated liquid was 585 ppm, whereas the water content in the recovered supernatant was reduced to 109.5 pp.

Example 6

[0104] The concentrated liquid (water content: 121.8 ppm) of the coenzyme Q10-producing microorganism was prepared in the same manner as in Example 1, cooled to 17 C., and subjected to separation so that the volume ratio of a slurry containing a solid and a filtrate became 1:9 by a separation method using Mitsubishi Dyna Filter (manufactured by Mitsubishi Kakoki Co., Ltd.) having one aluminum oxide disc with an average pore size of 0.2 gm and a filtration area of 0.034 square meters. As a result of analyses of the concentrated liquid and the filtrate, it was confirmed that the water content in the concentrated liquid was 121.8 ppm, whereas the water content in the filtrate was reduced to 67.1 ppm, the removal rate of ergosterol was 68.1%, and the purity of coenzyme Q10 was increased by 4.7% pt.

Example 7

[0105] The same concentrated liquid (water content: 121.8 ppm) of the coenzyme Q10-producing microorganism as that of Example 6 was cooled to 15 C., and subjected to separation so that the volume ratio of a slurry containing a solid and a filtrate became 1:9 by a separation method using Mitsubishi Dyna Filter (manufactured by Mitsubishi Kakoki Co., Ltd.). As a result of analyses of the filtrate, the water content was reduced to 54.7 ppm, the removal rate of ergosterol was 69.8%, and the purity of coenzyme Q10 was increased by 4.5% pt.

Example 8

[0106] The balance of coenzyme Q10 was measured from the coenzyme Q10 concentrations in the concentrated liquid of the coenzyme Q10-producing microorganism used in Example 7, the obtained filtrate, the slurry containing a solid after solid-liquid separation, and the washing solution of the apparatus. As a result, it was confirmed that 100% recovery of coenzyme Q10 was possible without a loss of coenzyme Q10 by the separation.

Example 9

[0107] The concentrated liquid (509.9 ppm) of the coenzyme Q10-producing microorganism was prepared in the same manner as in Example 1, cooled to 15 C., and subjected to separation so that the volume ratio of a slurry containing a solid and a filtrate became 1:2.6 by a separation method using Ceramic Rotary Filter (manufactured by Hiroshima Metal & Machinery Co., Ltd.,) having one aluminum oxide disc with an average pore size of 0.2 gm and a filtration area of 0.0334 square meters. As a result of analyses of the concentrated liquid and the filtrate, it was confirmed that the water content in the concentrated liquid was 509.9 ppm, whereas the water content in the filtrate was reduced to 133.2 ppm, the removal rate of ergosterol was 69.1%, and the purity of coenzyme Q10 was increased by 7.0% pt.

Example 10

[0108] To the extract obtained in the same manner as in Example 1 was added 0.5 vol % of the slurry containing a large amount of solid after the separation obtained in Example 7 relative to the extract, the mixture was brought into contact and mixed with an aqueous alkaline solution in the same manner as in Example 1 and then washed with water, and the aqueous layer and the extract were separated. The coenzyme Q10 concentration in the aqueous layer was 0.01 wt % or less, and thus there was no loss of coenzyme Q10. There were also no problems with the steps of alkali treatment and washing. From the above, it was confirmed that there is no problem in returning the slurry containing a large amount of solid to the step before being brought into contact and mixed with the aqueous alkaline solution.

Example 11

[0109] To the extract obtained in the same manner as in Example 1 was added 2 vol % of the slurry containing a large amount of solid after the separation obtained in Example 7 relative to the extract, the mixture was brought into contact and mixed with an aqueous alkaline solution in the same manner as in Example 1 and then washed with water, and the aqueous layer and the extract were separated. The coenzyme Q10 concentration in the aqueous layer was 0.01 wt % or less, and thus there was no loss of coenzyme Q10. There were also no problems with the steps of alkali treatment and washing. From the above, it was confirmed that there is no problem in returning the slurry containing a large amount of solid to the step before being brought into contact and mixed with the aqueous alkaline solution.

Example 12

[0110] Into the extract obtained by the same method as in Example 1 were poured 0.5 vol % of the slurry containing a large amount of solid after the separation obtained in Example 7 and 8 vol % of a 4 wt % aqueous solution of sodium hydroxide relative to the extract. The extract and the aqueous layer were separated in a settler, a continuous operation of continuously discharging the aqueous layer was conducted for 2 hours. Throughout the operation, ease of separation between the extract and the aqueous layer in the settler was satisfactory. The coenzyme Q10 concentration in the aqueous layer recovered after the completion of the operation was 0.002 wt %, and thus there was no loss of coenzyme Q10. There were also no problems with the above-described steps. From the above, it was confirmed that there is no problem in returning the concentrate containing a large amount of solid to the step before being brought into contact and mixed with the aqueous alkaline solution.

Comparative Example 1

[0111] The same concentrated liquid (water content: 663.4 ppm) of the coenzyme Q10-producing microorganism as that of Example 1 was placed in an environment of 40 C. for 2 hours and subjected to suction filtration in the same manner as in Example 1 to separate and remove a solid. As a result of analysis of the filtrate, it was confirmed that although the purity of coenzyme Q10 was increased by 3.2%pt., the water content in the filtrate was 334.7 ppm, and the removal rate of ergosterol was 2.9%.

Comparative Example 2

[0112] The same concentrated liquid (water content: 585 ppm) of the coenzyme Q10-producing microorganism as that of Example 5 was, under the condition of 30 C. without cooling, subjected to centrifugation at 9000 g for 5 minutes using the same centrifuge as that in Example 5 to recover a supernatant. It was confirmed that the water content in the recovered supernatant was reduced to 309.5 ppm.

Comparative Example 3

[0113] The concentrated liquid (water content: 550.5 ppm) of the coenzyme Q10-producing microorganism was prepared in the same manner as in Example 1. To the prepared extract were added 3.5 wt % of activated clay and 4 wt % of a filter aid (Rokahelp: manufactured by Mitsui Mining & Smelting Co., Ltd.), and the mixture was stirred at 40 C. and subjected to suction filtration to separate and remove a solid. For the filtration, a Kiriyama funnel and No. 5-C filter paper for use in the Kiriyama funnel were used. As a result, it was confirmed that the water content in the concentrated liquid was 550.5 ppm, whereas the water content in the filtrate was 402.5 ppm, the yield of coenzyme Q10 was 97.8%, the removal rate of ergosterol was 8.8%, and the purity improvement percent point of coenzyme Q10 was 0.5% pt.

Comparative Example 4

[0114] To the same concentrated liquid (water content: 550.5 ppm) of the coenzyme Q10-producing microorganism as that of Comparative Example 3 were added 3.5 wt % of activated clay and 4 wt % of a filter aid (Rokahelp: manufactured by Mitsui Mining & Smelting Co., Ltd.), and the mixture was stirred at 40 C. and then cooled to 18 C. Then, a solid was separated and removed in the same manner as in Comparative Example 3. As a result, it was confirmed that the water content in the filtrate was 220.5 ppm, the removal rate of ergosterol was 62.4%, and the purity of coenzyme Q10 was increased by 3.5% pt, but the yield of coenzyme Q10 was 98.4%.