Method for producing coenzyme Q10

Abstract

A method of producing coenzyme Q10 includes contacting an extract from a coenzyme Q10-producing microorganism with an adsorbent (A) such that the adsorbent (A) adsorbs a component of the extract other than coenzyme Q10, and that coenzyme Q10 is obtained. The adsorbent (A) includes aluminum silicate at a content of 50% or more.

Claims

1. A method of producing coenzyme Q10, the method comprising: contacting an extract comprising coenzyme Q10 from a coenzyme Q10-producing microorganism with an adsorbent (A), wherein the adsorbent (A) adsorbs components of the extract other than coenzyme Q10, and a solution of the coenzyme Q10 having a higher purity compared to the extract is obtained, wherein the adsorbent (A) comprises 50% or more of aluminum silicate.

2. The method of claim 1, further comprising: before or after contacting the extract with the adsorbent (A), contacting the extract with an adsorbent (B).

3. The method of claim 2, wherein the adsorbent (B) is at least one selected from the group consisting of an adsorbent comprising activated carbon, a synthetic adsorbent obtained by copolymerizing styrene and divinylbenzene, a phenol-formaldehyde resin adsorbent, an aldehyde adsorbent, magnesium silicate, aluminum hydroxide, aluminum oxide, magnesium oxide, diatomite, activated alumina, silica gel, silica-magnesia gel, an adsorbent obtained by chemically binding an alkyl group or an allyl group on an inorganic carrier, an aromatic adsorbent, a methacrylate ester adsorbent, synthetic zeolite, and a pearlite filter medium.

4. The method of claim 2, wherein the adsorbent (B) comprises a pearlite filter medium.

5. The method of claim 1, wherein the contacting comprises contacting the extract with a mixture comprising the adsorbent (A) and an adsorbent (B).

6. The method of claim 5, wherein the adsorbent (B) is at least one selected from the group consisting of, an adsorbent comprising activated carbon, a synthetic adsorbent obtained by copolymerizing styrene and divinylbenzene, a phenol-formaldehyde resin adsorbent, an aldehyde adsorbent, magnesium silicate, aluminum hydroxide, aluminum oxide, magnesium oxide, diatomite, activated alumina, silica gel, silica-magnesia gel, an adsorbent obtained by chemically binding an alkyl group or an allyl group on an inorganic carrier, an aromatic adsorbent, a methacrylate ester adsorbent, synthetic zeolite, and a pearlite filter medium.

7. The method of claim 5, further comprising: separating the extract of the coenzyme Q10-producing microorganism contacted with the adsorbent (A) and the adsorbent (B) from the adsorbent (A) and the adsorbent (B); and treating the solution comprising the coenzyme Q10 separated from the absorbent (A) and the adsorbent (B).

8. The method of claim 7, wherein the treating comprises conducting a condensation, a solvent exchange, an oxidation, a reduction, a column chromatography, a crystallization, or a combination thereof.

9. The method of claim 5, wherein a weight ratio of the adsorbent (A) to the adsorbent (B) in the mixture is from 40: 60 to 60: 40.

10. The method of claim 5, wherein a weight ratio of the adsorbent (A) to the adsorbent (B) in the mixture is 50: 50.

11. The method of claim 1, further comprising: separating the extract contacted with the adsorbent (A) from the adsorbent (A); and treating the solution comprising the coenzyme Q10 from the absorbent (A).

12. The method of claim 11, wherein the treating comprises conducting a condensation, a solvent exchange, an oxidation, a reduction, a column chromatography, and a crystallization, or a combination thereof.

13. The method of claim 1, wherein the adsorbent (A) comprises 60% or more of aluminum silicate.

14. The method of claim 1, wherein the adsorbent (A) comprises 65% or more of aluminum silicate.

15. The method of claim 1, wherein the aluminum silicate comprises from 50% to 70% of silicon dioxide.

16. The method of claim 1, wherein the aluminum comprises from 55% to 65% of silicon dioxide.

17. The method of claim 1, wherein the aluminum silicate comprises crystal water.

18. The method of claim 1, wherein the aluminum silicate comprises a solid acid.

19. The method of claim 1, wherein upon the contacting, the adsorbent (A) absorbs ergosterol and thereby removes ergosterol from the extract, wherein a removal rate of ergosterol is 30% or more.

20. The method of claim 1, wherein upon the contacting, the adsorbent (A) absorbs ergosterol and thereby removes ergosterol from the extract, wherein a removal rate of ergosterol is 40% or more.

Description

EXAMPLES

(1) 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.

(2) The yield of coenzyme Q10 was calculated by analyzing the coenzyme Q10 concentrations in the solutions before and after the adsorption treatment. The concentration of coenzyme Q10 was measured by high-performance liquid chromatography (HPLC) (manufactured by SHIMADZU) in the following condition.

(3) HPLC Measurement Condition Column: YMC-Pack ODS-A manufactured by YMC Oven temperature: 30 C. Mobile phase: methanol/hexane=85/15 by volume Flow rate: 1.0 ml/min Detection: UV 275 nm

(4) A purity improvement percent point 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 adsorption treatment.

(5) A removal rate of ergosterol was calculated by measuring ergosterol concentrations in the solutions before and after the adsorption treatment 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 concentration before the adsorption treatmentERG concentration after the adsorption treatment)/(ERG concentration before the adsorption treatment)}100

Example 1

(6) 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, extract malt 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 cells were dried by using a spray dryer. To the obtained dried microorganism, 1.5 times amount of hexane to the volume of the culture medium before the centrifugation was added. The mixture was stirred at 50 C. for 1 hour to extract coenzyme Q10. The extract of the coenzyme Q10-producing microorganism was concentrated so that the coenzyme Q10 concentration was adjusted to 25 g/L. The reduced coenzyme Q10 ratio in the concentrate, i.e. the ratio of reduced coenzyme Q10 in the total coenzyme Q10, was about 5 wt %. Into the concentrated extract, 50 wt % to the wt % of coenzyme Q10 of KYOWAAD 700 (manufactured by Kyowa Chemical Industry Co., Ltd., KW700SN) composed of synthetic aluminum silicate as the adsorbent (A) and KYOWAAD 200 (manufactured by Kyowa Chemical Industry Co., Ltd., KW200) composed of aluminum hydroxide as the adsorbent (B) were respectively added. The mixture was stirred at room temperature for 1 hour and then filtrated. The filtrate was analyzed; as a result, it was confirmed that the yield of coenzyme Q10 was 98.41%, the purity thereof was increased by 3.4 percent point, and the removal rate of ergosterol was 49.7%.

Example 2

(7) Into the concentrated extract of the coenzyme Q10-producing microorganism obtained similarly to Example 1, 50 wt % to the wt % of coenzyme Q10 of KYOWAAD 700 (manufactured by Kyowa Chemical Industry Co., Ltd., KW700SN) as the adsorbent (A) and pearlite filter medium Rokahelp (manufactured by MITSUI MINING & SMELTING CO., LTD.) as the adsorbent (B) were respectively added. The mixture was subjected to the adsorption treatment in a similar condition to Example 1. As a result, the yield of coenzyme Q10 was 98.97%, the purity thereof was increased by 2.8 percent point, and the removal rate of ergosterol was 54.7%.

Example 3

(8) Into the concentrated extract of the coenzyme Q10-producing microorganism obtained similarly to Example 1, 50 wt % to the wt % of coenzyme Q10 of KYOWAAD 700 (manufactured by Kyowa Chemical Industry Co., Ltd., KW700SN) as the adsorbent (A) and a solid solution of aluminum oxide and magnesium oxide KYOWAAD 2000 (manufactured by Kyowa Chemical Industry Co., Ltd., KW2000) as the adsorbent (B) were respectively added. The mixture was subjected to the adsorption treatment in a similar condition to Example 1. As a result, the yield of coenzyme Q10 was 96.26%, the purity thereof was increased by 4.4 percent point, and the removal rate of ergosterol was 37.6%.

Example 4

(9) The microorganism culture medium which was obtained by the cultivation similarly to Example 1 and which contained coenzyme Q10 was concentrated by centrifugation, and the supernatant was separated to be removed. The obtained heavy layer was subjected to cell disruption under pressure of about 100 MPa to obtain concentrated microorganism culture medium. Into the culture medium, hexane and isopropanol were added as extraction solvents to extract coenzyme Q10. The extract of the coenzyme Q10-producing microorganism was concentrated so that the coenzyme Q10 concentration was adjusted to 25 g/L. Into the extract, 9 wt % to the wt % of coenzyme Q10 of KYOWAAD 700 (manufactured by Kyowa Chemical Industry Co., Ltd., KW700SN) as the adsorbent (A) was added. The mixture was stirred at room temperature for 1 hour and then filtrated. The filtrate was analyzed; as a result, the yield of coenzyme Q10 was 93.83%, the purity thereof was increased by 3.3 percent point, and the removal rate of ergosterol was 2.8%.

Example 5

(10) Into the concentrated extract of the coenzyme Q10-producing microorganism obtained similarly to Example 1, 50 wt % to the wt % of coenzyme Q10 of KYOWAAD 700 (manufactured by Kyowa Chemical Industry Co., Ltd., KW700SN) as the adsorbent (A) and similarly synthetic aluminum silicate Nikkagel S (manufactured by Toshin Chemicals Co., Ltd.) were respectively added. The mixture was subjected to the adsorption treatment in a similar condition to Example 1. As a result, the yield of coenzyme Q10 was 95.17%, the purity thereof was increased by 2.9 percent point, and the removal rate of ergosterol was 39.4%.

Example 6

(11) 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, extract malt 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 cells were dried by using a spray dryer. To the obtained dried microorganism, 1.5 times amount of ethyl acetate to the volume of the culture medium before the centrifugation was added. The mixture was stirred at 50 C. for 1 hour to extract coenzyme Q10. The extract of the coenzyme Q10-producing microorganism was concentrated so that the coenzyme Q10 concentration was adjusted to 25 g/L. The reduced coenzyme Q10 ratio in the concentrate was about 5 wt %. Into the concentrated extract, 50 wt % to the wt % of coenzyme Q10 of KYOWAAD 700 (manufactured by Kyowa Chemical Industry Co., Ltd., KW700SN) as the adsorbent (A) and Rokahelp (manufactured by MITSUI MINING & SMELTING CO., LTD.) as the adsorbent (B) were respectively added. The mixture was stirred at room temperature for 1 hour and then filtrated. The filtrate was analyzed; as a result, it was confirmed that the yield of coenzyme Q10 was 94.11%, the purity thereof was increased by 5.4 percent point, and the removal rate of ergosterol was 38.2%.

Comparative Example 1

(12) Into the concentrated extract of the coenzyme Q10-producing microorganism obtained similarly to Example 1, 50 wt % to the wt % of coenzyme Q10 of KYOWAAD 200 (manufactured by Kyowa Chemical Industry Co., Ltd., KW200) and Rokahelp (manufactured by MITSUI MINING & SMELTING CO., LTD.) were respectively added. The mixture was subjected to the adsorption treatment in a similar condition to Example 1. As a result, it was confirmed that the yield of coenzyme Q10 was 96.46%, the purity thereof was merely increased by 1.5 percent point, and the removal rate of ergosterol was 10.7%.

Comparative Example 2

(13) Into the concentrated extract of the coenzyme Q10-producing microorganism obtained similarly to Example 1, 50 wt % to the wt % of coenzyme Q10 of Rokahelp (manufactured by MITSUI MINING & SMELTING CO., LTD.) was added. The mixture was subjected to the adsorption treatment in a similar condition to Example 1. As a result, the yield of coenzyme Q10 was 94.05%, the purity thereof was increased by 0 percent point, and the removal rate of ergosterol was 2.0%.

Comparative Example 3

(14) Into the concentrated extract of the coenzyme Q10-producing microorganism obtained similarly to Example 1, 50 wt % to the wt % of coenzyme Q10 of KYOWAAD 2000 (manufactured by Kyowa Chemical Industry Co., Ltd., KW2000) and Rokahelp (manufactured by MITSUI MINING & SMELTING CO., LTD.) were respectively added. The mixture was subjected to the adsorption treatment in a similar condition to Example 1. As a result, it was confirmed that the yield of coenzyme Q10 was 82.82%, the purity thereof was merely increased by 1.8 percent point, and the removal rate of ergosterol was 4.1%.

Comparative Example 4

(15) Into the concentrated extract of the coenzyme Q10-producing microorganism obtained similarly to Example 1, 50 wt % to the wt % of coenzyme Q10 of magnesium silicate KYOWAAD 600 (manufactured by Kyowa Chemical Industry Co., Ltd., KW600) and Rokahelp (manufactured by MITSUI MINING & SMELTING CO., LTD.) were respectively added. The mixture was subjected to the adsorption treatment in a similar condition to Example 1. As a result, it was confirmed that the yield of coenzyme Q10 was 96.29%, the purity thereof was merely increased by 1.3 percent point, and the removal rate of ergosterol was 4.2%.

Comparative Example 5

(16) Into the concentrated extract of the coenzyme Q10-producing microorganism obtained similarly to Example 1, 50 wt % to the wt % of coenzyme Q10 of activated carbon and Rokahelp (manufactured by MITSUI MINING & SMELTING CO., LTD.) were respectively added. The mixture was subjected to the adsorption treatment in a similar condition to Example 1. As a result, it was confirmed that the yield of coenzyme Q10 was 89.99%, the purity thereof was merely increased by 0.3 percent point, and the removal rate of ergosterol was 29.7%.

Comparative Example 6

(17) Into the concentrated extract of the coenzyme Q10-producing microorganism obtained similarly to Example 1, 50 wt % to the wt % of coenzyme Q10 of activated alumina 300 (manufactured by NACALAI TESQUE, INC.) and Rokahelp (manufactured by MITSUI MINING & SMELTING CO., LTD.) were respectively added. The mixture was subjected to the adsorption treatment in a similar condition to Example 1. As a result, it was confirmed that the yield of coenzyme Q10 was 96.78%, the purity thereof was merely increased by 1.6 percent point, and the removal rate of ergosterol was 1.1%.

(18) TABLE-US-00001 TABLE 1 Purity improvement Ergosterol percent removal Raw material Yield point rate for extraction Solvent Adsorbent (A) Adsorbent (B) (%) (%) (%) Example 1 non-disrupted hexane KW700SN (50 wt. %) KW200 98.41 3.4 49.7 dried cell (50 wt %) Example 2 non-disrupted hexane KW700SN (50 wt %) Rokahelp 98.97 2.8 54.7 dried cell (50 wt %) Example 3 non-disrupted hexane KW700SN (50 wt %) KW2000 96.26 4.4 37.6 dried cell (50 wt %) Example 4 heavy layer hexane KW700SN (9 wt %) 93.83 3.3 2.8 after disruption (iPrOH) Example 5 non-disrupted hexane KW700SN (50 wt %) 95.17 2.9 39.4 dried cell Nikkagel S (50 wt %) Example 6 non-disrupted ethyl KW700SN (50 wt %) Rokahelp 94.11 5.4 38.2 dried cell acetate (50 wt %) Comparative non-disrupted hexane KW200 Rokahelp 96.46 1.5 10.7 example 1 dried cell (50 wt %) (50 wt %) Comparative non-disrupted hexane Rokahelp 94.05 0.0 2.0 example 2 dried cell (50 wt %) Comparative non-disrupted hexane KW2000 Rokahelp 82.82 1.8 4.1 example 3 dried cell (50 wt %) (50 wt %) Comparative non-disrupted hexane KW600 Rokahelp 96.29 1.3 4.2 example 4 dried cell (50 wt %) (50 wt %) Comparative non-disrupted hexane activated Rokahelp 89.99 0.3 29.7 example 5 dried cell carbon (50 wt %) (50 wt %) Comparative non-disrupted hexane activated Rokahelp 96.78 1.6 1.1 example 6 dried cell alumina (50 wt %) (50 wt %)

(19) The present application addresses improvement for easy and stably mass production of coenzyme Q10 at a low cost in the above-described conventional methods.

(20) For example, with respect to the method of Patent document 1, when a large amount of impurity coexists, such as the case of an extract of coenzyme Q10-producing microorganism, it is difficult to obtain coenzyme Q10 having a high purity by crystallization only. Even when highly-pure coenzyme Q10 can be obtained, it is necessary to strictly control the operating condition such as crystallization operating temperature and the time required for the crystallization process is prolonged. With respect to the adsorption method of Patent document 2, since the purpose of the method is to adsorb coenzyme Q10 itself on an adsorbent, the step of separating and eluting coenzyme Q10 from the adsorbent by using an elution solvent is required after the adsorption treatment to obtain coenzyme Q10.

(21) An aspect of the present invention to solve the above-described problems is to provide a method for stably producing coenzyme Q10 with a simple coenzyme Q10 production step by efficiently removing impurity of a microorganism from an extract of coenzyme Q10-producing microorganism.

(22) The inventors of the present invention intensively studied for solving the above problem. As a result, the present inventors completed the present invention by finding that the specific adsorbent is useful for efficiently purifying coenzyme Q10, since the adsorbent hardly adsorbs coenzyme Q10 itself but selectively adsorbs a component derived from cells of a coenzyme Q10-producing microorganism except for coenzyme Q10.

(23) The present invention relates to a method for producing coenzyme Q10 characterized in comprising the step of contacting an extract of a coenzyme Q10-producing microorganism with an adsorbent (A), wherein a main component of the adsorbent (A) is aluminum silicate.

(24) The present invention preferably relates to the above-described method wherein 50% or more of the adsorbent (A) is aluminum silicate.

(25) The present invention preferably relates to the above-described method wherein an adsorbent (B) is used in addition to an adsorbent (A).

(26) The present invention preferably relates to the above-described method wherein the adsorbent (B) is one or more selected from the group consisting of activated carbon, an adsorbent containing activated carbon as a main component, a synthetic adsorbent obtained by copolymerizing styrene and divinylbenzene, a phenol-formaldehyde resin adsorbent, an aldehyde adsorbent, magnesium silicate, aluminum hydroxide, aluminum oxide, magnesium oxide, silica gel, silica-magnesia gel, an adsorbent obtained by chemically binding an alkyl group or an allyl group on an inorganic carrier, diatomite, activated alumina, an aromatic adsorbent, a methacrylate ester adsorbent, synthetic zeolite, and a pearlite filter.

(27) The present invention preferably relates to the above-described method, comprising the steps of separating the extract of the coenzyme Q10-producing microorganism contacted with the adsorbent (A) from the adsorbent (A) and further treating the separated extract.

(28) The present invention preferably relates to the above-described method wherein the step of treating is one or more steps selected from a condensation, a solvent exchange, an oxidation, a reduction, a column chromatography and a crystallization.

(29) According to the embodiments of the present invention, high quality coenzyme Q10 can be obtained successfully in terms of workability and economy with easily adsorbing impurity to be removed and without adsorbing coenzyme Q10 only by contacting a solution containing coenzyme Q10 with an adsorbent (A) having aluminum silicate as a main component.

(30) In addition, an impurity removal efficiency can be further improved by using an adsorbent (B) different from the adsorbent (A) in addition to the adsorbent (A).

(31) Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.