Title of Invention	A PROCESS FOR PRODUCING THE OXIDIZED COENZYME Q10
Abstract	The present invention discloses a process for producing the oxidized coenzyme Q10 which comprises culturing reduced coenzyme Q10 producing microorganisms in a culture medium containing a carbon source, a nitrogen source, a phosphorus source and a micronutrient to obtain microbial cells containing reduced coenzyme Q10 at a ratio of not less than 70 mole % among the entire coenzymes Q10, optionally disrupting the microbial cells; and either oxidizing thus-produced reduced coenzyme Q10 to oxidized coenzyme Q10 using an oxidizing agent and then extracting the resultant by an organic solvent, or extracting thus-producing reduced coenzyme Q10 by an organic solvent, purifying optionally and oxidizing the resultant to oxidized coenzyme Q10 using an oxidizing agent.

Title of Invention

A PROCESS FOR PRODUCING THE OXIDIZED COENZYME Q10

Abstract

The present invention discloses a process for producing the oxidized coenzyme Q10 which comprises culturing reduced coenzyme Q10 producing microorganisms in a culture medium containing a carbon source, a nitrogen source, a phosphorus source and a micronutrient to obtain microbial cells containing reduced coenzyme Q10 at a ratio of not less than 70 mole % among the entire coenzymes Q10, optionally disrupting the microbial cells; and either oxidizing thus-produced reduced coenzyme Q10 to oxidized coenzyme Q10 using an oxidizing agent and then extracting the resultant by an organic solvent, or extracting thus-producing reduced coenzyme Q10 by an organic solvent, purifying optionally and oxidizing the resultant to oxidized coenzyme Q10 using an oxidizing agent.

Full Text	THIS APPLICATION HAS BEEN DIVIDED OUT OF INDIAN APPLICATION NO. 00918/KOLNP/2004 TECHNICAL FIELD The present invention relates to a process for producing the reduced coenzyme Qio represented by the following formula (I): More particularly, the present invention relates to a process for producing reduced coenzyme Q10 which comprises culturing reduced coenzyme Q10- producing microorganisms to obtain microbial cells containing reduced coenzyme Q10 at a ratio of not less than 70 mole % among the entire coenzymes Q10, optionally disrupting the microbial cells and recovering thus-produced reduced coenzyme Q10. The present invention also relates to a process for producing oxidized coenzyme Q10 which comprises either recovering oxidized coenzyme Q10 after oxidizing the above- nentioned microbial cells or disrupted product thereof, or recovering reduced coenzyme Q10 from the above-mentioned nicrobial cells or disrupted product thereof to oxidize thus-obtained reduced coenzyme Q10 thereafter. BACKGROUND ART The reduced coenzyme Q10 (I) and the oxidized coenzyme Q10 (II) are mitochondrial electron transport system-constituting factors in cells of a living body of luman and deal with ATP production by working as electron carriers in oxidative phosphorization reactions. Conventionally, oxidized coenzyme Q10 has been widely ised for supplementary nutrient foods and cosmetic products in addition to pharmaceutical products as a pharmaceutically and physiologically effective substance for a variety of diseases. On the other hand, reduced coenzyme Q10 has not so nuch drawn attention so far; however, in these years, there las been reported that reduced coenzyme Q10 is more affective in various applications than oxidized coenzyme Q10. For example, japanise Kukai Publication Hei-10-330251 discloses an antihypercholesterolemia agent having axcellent cholesterol reducing function, an iritihyperlipeuiia agent, and an agent for curing and preventing arteriosclerosis which contain reduced coenzyme Qio as an active ingredient. In addition, Japanese Kokai Publication Hei-10-109933 discloses a pharmaceutical composition excellent in oral absorbability comprising coenzyme Q10 including reduced coenzyme Q10 as an active ingredient. Furthermore, reduced coenzyme Q10 is effective as an antioxidant and a radical scavenger. R. Stocker, et al. nave reported that reduced coenzyme Qio prevented peroxidation of human LDL more efficiently than a- tocopherol, lycopene and (3-carotene (Proceedings of the NFational Academy of Science of the United States of America, vol. 88, pp. 1646-1650, 1991) . It has been known that oxidized coenzyme Q10 and reduced coenzyme Q10 are in a certain type of equilibrium in a living body and that oxidized coenzyme Q10/ reduced coenzyme Qio absorbed in the living body are mutually reduced/oxidized. Reduced coenzyme Q10 is supposedly produced by a chemical synthesis method, similarly to the process for producing oxidized coenzyme Q10. But the synthesis process is supposed to be complicated, risky and costly. Moreover, in the case of chemical synthesis methods, it will be necessary to minimize the subgeneraLion and contamination of a (Z)-isomer, which is suspiciously unsafe (Biomedicai and Clinical Aspects of Coenzyme Q, vol. 3, pp. 19-30, 1981) . Europe Pharmacopoeia regulates that a content of (Z)-isomer in oxidized coenzyme Q10 must be not more than 0.1%. As another process for producing reduced coenzyme Q10, it can be supposed a method of utilizing microbial cells, that is, a method for separating and recovering reduced coenzyme Q10 from reduced coenzyme Q10-producing microorganisms. However, the reduced coenzyme Q10 produced by the microbial cells of the above mentioned microorganisms contains a large amount of oxidized coenzyme Q10, and the separation and recovery of reduced coenzyme Q10 by a conventional method results in high cost. The following are documents describing the presence of reduced coenzyme Q10 in microbial cells and there have been known the following examples of bacteria. (1) An example describing that at lowest 5 to 10% by weight and at highest 30 to 60% by weight of reduced coenzyme Q10 are present among the entire coenzymes Q10 in culture cells of photosynthesis bacteria (Japanese Kokai Publication Sho- 57-70834). (2) An example describing that the genus Pseudomonas is subjected to thermal extraction by an organic solvent in the presence of sodium hydroxide and pyrogallol, and the resultant is treated with 5% sodium hydrosulfite solution, and further dehydrated and concentrated to collect an acetone-soluble portion, and an oil containing reduced coenzyme Q10 is obtained (Japanese Kokai Publication Sho- 60-75294). Both of the above (1) and (2) aim to convert a mixture of the obtained reduced coenzyme Q10 and oxidized coenzyme Q10 or the obtained reduced coenzyme Q10 into oxidized coenzyme Q10 by further oxidation. Thus, reduced coenzyme Q10 is only described as an intermediate substance in producing oxidized coenzyme Q10. In the above (1), photosynthesis bacteria are used, the culture of which is complicated. Furthermore, in the microbial cells of the above-mentioned microorganisms, when the production of reduced coenzyme Q10 is aimed at, it cannot be said that the ratio of reduced coenzyme Q10 among the entire coenzymes Q10 is sufficient. The above (2) comprises a process of converting oxidized coenzyme Q10 contained in a hexane phase into reduced coenzyme Q10 by sodium hydrosulfite, a reducing agent (see Example 3 in Japanese Kokai Publication Sho-60- 75294). Thus, the ratio of reduced coenzyme Q10 among the entire coenzymes Q10 in the microbial cells is not clear. Furthermore, in both of the above (1) and (2), the production amount of coenzymes Q in culture are not described. As described above, microbial cells containing reduced coenzyme Q10 at high ratio have not been reported yet. Still less, it has not been known a fermentation production of reduced coenzyme Q10 on the industrial scale, that is, a method comprising culturing microorganisms to obtain microbial cells containing reduced coenzyme Q10 at high ratio among the entire coenzymes Q10, and recovering reduced coenzyme Q10 to obtain high-purity reduced coenzyme Q10. Under such circumstances, if a method for obtaining a large quantity of coenzyme Q10 containing reduced coenzyme Q10 at high ratio by culturing microorganisms ' is found, it can be a highly useful method for producing reduced coenzyme Q10. SUMMARY OF THE INVENTION It is an object of the present invention to provide a process for producing reduced coenzyme Q10 safely and efficiently on the industrial scale by culturing reduced coenzyme Q10-producing microorganisms for obtaining microbial cells containing reduced coenzyme Q10 at high ratio and suitably recovering reduced coenzyme Q10 from the microbial cells. It is another object of the present invention to provide a process for producing oxidized coenzyme Q10 in simple processes by culturing reduced coenzyme Q10- producing microorganisms for obtaining microbiai cells containing reduced coenzyme Q10 at high ratio, and oxidizing the reduced coenzyme Q10 obtained from the microbiai cells as an intermediate substance in producing oxidized coenzyme Q10. That is, the present invention relates to a process for producing the reduced coenzyme Q10 represented by the following formula (I): which comprises culturing reduced coenzyme Q10- producing microorganisms in a culture medium containing a carbon source, a nitrogen source, a phosphorus source and a micronutrient to obtain microbial cells containing reduced coenzyme Q10 at a ratio of not less than 70 mole % among the entire coenzymes Q10, optionally disrupting the microbial cells and extracting thus-produced reduced coenzyme Q10 by an organic solvent. Furthermore, the present invention also relates to a process for producing the oxidized coenzyme Q10 represented by the following formula (II): which comprises culturing reduced coenzyme Q10- producing microorganisms in a culture medium containing a carbon source, a nitrogen source, a phosphorus source and a micronutrient to obtain microbial cells containing reduced coenzyme Q10 at a ratio of not less than 70 mole % among the entire coenzymes Q10, optionally disrupting the microbial cells; and either oxidizing thus-produced reduced coenzyme Q10 to oxidized coenzyme Q10 and then extracting the resultant y an organic solvent, or extracting thus-produced reduced coenzyme Q10 by an organic solvent, purifying optionally md oxidizing the resultant to oxidized coenzyme Q10. According to the processes of the present invention, ceduced coenzyme Q10 can be produced cheaply on the industrial scale by considerably simple steps comprising culturing microorganisms and recovering reduced coenzyme Q10 In addition, oxidized coenzyme Q10 can also be produced by simple processes. Moreover, these coenzymes Q10 produced by microorganisms basically do not contain (Z)- isomers thereof, and (all-E) isomers thereof can be obtained, which are same as those contained in meat, fish, etc. DETAILED DISCRIPTION OF THE INVENTION In the present invention, at first, reduced coenzyme Q10-producing microorganisms are cultured to obtain microbial cells containing reduced coenzyme Q10 at a ratio of not less than 70 mole %, preferably not less than 75 mole %, among the entire coenzymes Q10 (fermentation). The microbial cells containing reduced coenzyme Q10 aL such high ratio among the entire coenzymes Q10 can be basically obtained by culturing microorganisms capable of producing reduced coenzyme Q10 at a ratio of not less than 7 0 mole %, preferably not less than 7 5 mole %, among the entire coenzymes Q10. How much ratio the microorganisms can produce reduced coenzyme Q10 among the entire coenzymes Q10 can be evaluated, for example, by a method comprising culturing the microorganisms with shaking (amplitude: 2 cm, 310 reciprocation/min) at 25°C for 72 hours in 10 mL of a culture medium [(glucose: 20 g, peptone: 5 g, yeast extract: 3g, malt extract: 3 g)/L, pH: 6.0] using a test tube (inner diameter: 21 mm, entire length: 200 mm). Although the preferable culture conditions for the fermentation production on the industrial scale will be iescribed later, the above-mentioned culture condition is one method for standardizing the ratio of reduced coenzyme Q10 produced, which microorganisms have as its ability, so as to reflect the ratio within the range without having significant inaccuracies. Under the above-mentioned culture condition, it is preferable to use microbial cells wherein a content of reduced coenzyme Q10 is at a ratio of not less than 70 mole %, preferably not less than 75 mole %, among the entire coenzymes Q10, for the present invention. It is still more preferable to use microorganisms having a productivity of reduced coenzyme Q10 per unit culture medium of generally not less than 1 g/mL, preferably not less than 2 (.g/mL under the above-mentioned culture condition. The above-mentioned content of reduced coenzyme Q10 and ratio of reduced coenzyme Q10 among the entire coenzymes Q10 can be confirmed by physically disrupting the microbial cells, extracting coenzyme Q10 from thus-obtained cells by an organic solvent and performing HPLC analysis. Specifically, the measurement can be carried out according to the following procedures: (1) The broth of microorganism is optionally concentrated, 10 parts by volume of the broth are displaced to a screw cap test tube (inner diameter: 16.5 mm, entire length: 130 mm), and 10 parts by volume of glass beads are added (425 to 600 urn, manufactured by SIGM7A Co.); (2) 3 parts by volume of isopropanol and 18.5 parts by volume of n-hexane relative to 10 parts by volume of the broth are added under a nitrogen atmosphere; (3) microbial cell disruption and extraction are carried out by vigorously shaking of the mixture for 3 minutes under a nitrogen atmosphere; and (4) the obtained hydrophobic organic solvent phase (n- hexane phase) is evaporated (bath temperature: 40°C) under reduced pressure to analyze the resultant by HPLC. Column: YMC-Pack 4.6 x 250 mm (manufactured by YMC. Co., Ltd.) Mobile phase: methanol/n-hexane = 85/15 Flow rate: 1 mL/min, Detection: UV 275 nm Retention time: reduced coenzyme Q10 13.5 min oxidized coenzyme Q10 22.0 min The above-mentioned measurement method is provided for the obtained result to reflect the reduced coenzyme Q10 content and the ratio of reduced coenzyme Q10 among the entire coenzymes Q10 as accurate as possible, and to standardize the content and the ratio of reduced coenzyme Q10, which can be guaranteed at the minimum. This method has been demonstrated, by several experimentations performed by the present inventors, easy and suitable to be carried out. As the above-mentioned reduced coenzyme Q10-producing microorganisms to be used in the present invention, bacteria, yeast and fungi may be used without any specific limitation. As specific examples of the above-mentioned microorganisms, there may be mentioned, for example, microorganisms of the genus Agrobacterium, the genus Aspergillus, the genus Acetobacter, the genus Aminobacter, the genus Agromonas, the genus Acidiphilium, the genus Bulleromyces, the genus Bullera, the genus Brevundimonas, the genus Cryptococcus, the genus Chionosphaera, the genus Candida, the genus Cerinosterus, the genus Exisophiala, the genus Exobasidium, the genus Fellomyces, the genus Filobasidiella, the genus Filobasidium, the genus Geotrichum, the genus Graphiola, the genus Gluconobacter, the genus Kockovaella, the genus Kurtzmanomyces, the genus Lalaria, the genus Leucosporidium, the genus Legionella, the genus Methylobacterium, the genus Mycoplana, the genus Dosporidium, the genus Pseudomonas, the genus Psedozyma, the genus Paracoccus, the genus Petromyc, the genus Rhodotorula, the genus Rhodosporidium, the genus Rhizomonas, the genus Rhodobium, the genus Rhodoplanes, the genus Rhodopseudomonas, the genus Rhodobacter, the genus Sporobolomyces, the genus Sporidiobolus, the genus Saitoella/ the genus Schizosaccharomyces, the' genus Sphingomonas, the genus Sporotrichum, the genus Sympodiomycopsis, the genus Sterigmatosporidium, the genus Tapharina. the genus Tremella, the genus Trichosporon, the genus Tilletiaria, the genus Tilletia, the genus Tolyposporium, the genus Tilletiopsis, the genus Ustilago, fhp genus Udeniomyce, the genus Xanthophllomyces, the genus Xanthobacter, the genus Paecilomyces, the genus Acremonium, the genus Hyhomonus, and the genus Rhizobium. In terms of the culture easiness and productivity, bacteria (preferably nonphotosynthetic bacteria) and yeast are preferred. As the bacteria, there may be mentioned, for example, the genus Agrobacterium, the genus Gluconobacter and the like. As the yeast, there may be mentioned, for example, the genus Schizosaccharomyces, the genus Saitoeiia and the like. As preferable species, there may be mentioned, for example, Agrobacterium tumefacience IFO13263, Agrobacterium radiobacter ATCC4718, Aspergiiius ciavatus JCM1718, Acetobacter xylinum IFO15237, Aminobacter aganouensis JCM7854, Agromonas oligotrophica JCM1494, Acidiphilium multivorum JCM8 8 67, Bulleromyces albus IFO1192, Bullera armeniaca IFO10112, Brevundimonas diminuta JCM2788, Cryptococcus laurentii IFO0609, Chionosphaera apobasidialis CBS7430, Candida curvata ATCC10567, Cerinosterus luteoalbus JCM2923, Exisophiala alcalophila JCM12519, Exobasidium gracile IFO7788, Fellomyces fuzhouensis IFO10374, Filobasidiella neoformans CBS132, Filobasidium capsuloigenum CBS1906, Geotrichum capitatum JCM6258, Graphiola cylindrica IFO6426, Gluconobacter suboxydans IFO32 57, Kockovaella imperatae JCM7 82 6, Kurtzmanomyces nectairei IFO10118, Lalaria cerasi CBS275.28, Leucosporidium scottii IFO1212, Legionella anisa JCM7573, Methylobacterium extorguens JCM2 8 02, Mycoplana ramosa JCM7822, Oosporidium margaritiferum CBS2531, Pseudomonas denitrificans IAM 12023, Psaudomonas shuylkil.liensis IAM 1092, Psedozyma aphidis CBS517.23, Paracoccus denitrificans JCM6892, Petromyces alliaceus IFO7538, Rhodotorula glutinis IFO1125. Rhodotorula minuta IFO03 87, Rhodosporidium diobovatum ATCC1830, Rhizomonas suberifaciens IFO15212, Rhodobium orients JCM9337, Rhodoplanes elegans JCM922 4, Rhodopseudomonas palustris JCM2524, Rhodobacter capsulatus SB1003, Sporobolomyces holsaticus IFO1034, Sporobolomyces pararoseus IFO0471, Sporidiobolus johnsonii IFO1840, Saitoella complicata IFO10748, Schizosaccharomyces pombe IFO0347, Sphingomonas parapaucimobilis IFO15100, Sporotrichum cellulophilium ATCC2 04 93, Sympodiomycopsis paphiopedili JCM8318, Sterigmatosporidium polymorphum IFO10121, Sphingomonas adhesiva JCM7370, Tapharina caerulescens CBS351.35, Tremella mesenterica ATCC24438, Trichosporon cutaneum IFO1198, Tilletiaria anomaia CBS4 3 6.72, Tilletia caries JCM17 61, Tolyposporium buiiatum JCM2006, Tilletiopsis washintonesis CBS544, Ustilago esculenta IFO9887, Udeniomyces megalosporus JCM5269, Xanthophllomyces dendrorhous IFO10129, Xanthobacter flavus JCM1204, Paecilomyces lilacinus ATCC10114, Acremonium chrysogenum ATCC11550, Hyphomonas hirschiana ATCC33886, Rhizobium meliloti ATCC9930, and the like. As the reduced coenzyme Q10-producing microorganisms, not only the wild species of the above-mentioned microorganisms but also microorganisms in which the transcription and translation activities of the genes relevant to the biosynthesis of reduced coenzyme Q10 in the above-mentioned microorganisms, or the enzyme activity of the expressed protein are modified or improved can be used preferably, for example. As the means for modifying or improving the transcription and translation activities of the genes or the enzyme activity of the expressed protein, there may be mentioned gene recombination (including gene improvement, amplification and destruction by itself, external gene introduction, and gene improvement and proliferation of thus-introduced external genes) and mutagenesis by mutagens In particular, the mutagenesis by mutagens is preferred. The more preferable microorganisms usable for the present invention are microorganisms containing reduced coenzyme Q10 at a ratio of not less than 70 mole %, preferably not less than 75 mole %, more preferably not less than 80 mole %, still more preferably not less than 85 mole %, and particularly preferably not less than 90 mole %, among the entire coenzymes Q10 in the case where the above- mentioned modified or improved microorganisms, preferably microorganisms mutated by mutagens, are evaluated by the above-mentioned proliferation method and the measurement method. In the fermentation production on the industrial scale, it is preferable to use microorganisms haviny a productivity of reduced coenzyme Q10 per unit culture medium of not less than 1 ug/mL, preferably not less than 2 (.g/mL, more preferably not less than 3 \|g/mL, still more preferably not less than 5 .g/mL, particularly preferably not less than 10 g/mL, much more preferably not less than 15 g/mL, and most preferably not less than 20 ug/mL. The mutagenesis may be carried out by a single mutagenesis; however, mutagenesis is preferably carried out not less than 2 times. That is because it was found that the productivity of reduced coenzyme Q10 can be improved in the respective mutagenesis steps. It is needless to say that the candidates of the microbial cells to be mutated are, generally, those having a productivity of reduced coenzyme Q10 as high as possible in the case where the evaluation is carried out by the above-mentioned proliferation method and measurement method. The mutagenesis can be carried out by using optional and proper mutagens. The term "mutagen" encompasses, in a board definition, not only chemical agents having mutagenesis effects, for example, but also treatments such as UV radiation having mutagenesis effects. As examples of proper mutangens, there may be mentioned ethyl methanesulfonate, UV radiation, N-methyl-N'-nitro-N- nitrosoguanidine, nucleotide base analogues such as bromouracil, and acridines; however, they are not limited to these examples. According to a conventional mutagenesis technique, successively to the mutagenesis, a proper selection of microbial cells having high productivity of reduced coenzyme Q10 is carried out. For that, the culture obtained from a single colony should be evaluated, for example, by the above-mentioned proliferation method and measurement method. Since a reduced coenzyme Qlo crystal forms a white solid layer or a colorless liquid phase, a productivity of reduced coenzyme Q10 can be suitably evaluated by the above-mentioned measurement method at the time of selection of the colony. In the processes of the present invention, high productivity of reduced coenzyme Qlo in the fermentation production on the industrial scale can be achieved partially by using the microbial cells containing reduced coenzyme Q10 at a ratio of not less than 70 mole % among the entire coenzymes Q10 and, partially, by using the suitable conditions of culture (fermentation) for increasing a productivity of reduced coenzyme Q10 per unit culture medium as described below. It is particularly preferable to combinedly use suitable microbial cells described above and the suitable conditions of culture (fermentation) as described below. The culture is carried out, in general, in a culture medium containing major nutrients and micronutrients suited for microorganism proliferation. As the above-mentioned nutrients, there may be mentioned, for example, carbon sources (e.g. hydrocarbons such as glucose, sucrose, maltose, starch, corn syrup and molasses; alcohols such as methanol and ethanol), nitrogen sources (e.g. corn steep liquor, ammonium sulfate, ammonium phosphate, ammonium hydroxide, urea and peptone), phosphorus sources (e.g. ammonium phosphate and phosphoric acid) and micronutrients (e.g. minerals such as magnesium, potassium, zinc, copper, iron, manganese, molybdenum, sulfuric acid and hydrochloric acid; vitamins such as biotin, desthiobiotin and vitamin Bl; amino acids such as alanine and histidine; and natural raw materials containing vitamins such as yeast extract and malt extract); however, these are not limitative ones, and commonly used ones may be used. Incidentally, in natural components of a culture medium, such as yeast extract, phosphorus sources such as phosphates are contained. The above-mentioned nutrients can be appropriately used in combination. The culture is generally carried out at a temperature range of 15 to 45°C, preferably 20 to 37°C. If it is below 15°C, the proliferation speed of microorganisms tends to be too slow to allow the industrial production and at high temperatures exceeding 45°C, the viability of microorganisms tends to be easily hindered. In general, the culture is carried out at a pH range of 4 to 9, preferably 5 to 8 . If the pH is not more than 3 or not less than 10, proliferation of microorganisms tends to be easily inhibited. In the fermentation production on the industrial scale, although it depends on the microorganism species, the concentration of the carbon sources (including the produced alcohols) during the culture is preferably controlled to a concentration that no adverse effects are substantially caused on the productivity of reduced coenzyme Q10. Accordingly, it is preferable to control the culture so as to have the concentration of the carbon sources that no adverse effects are substantially caused on the productivity of reduced coenzyme Q10, that is, generally to not more than 20 g/L, preferably not more than 5 g/L, and more preferably not more than 2 g/L in the broth. To control the concentration of the carbon sources, a fed batch culture method is preferably used. The carbon source concentration in the broth can be controlled by adjusting the supply of nutrient sources (especially carbon sources) based on the culture control indexes such as pll, the dissolved oxygen concentration (DO) or the remaining saccharide concentration. Although it depends on the microorganism species, the supply of the nutrient sources nay be started from the initial stage of the culture or during the culture. The supply of the nutrient sources may be continuous or intermittent. Incidentally, in supplying the nutrient sources, it is preferable to supply the above- mentioned carbon sources Lo the culture medium separately from other components. The culture can be completed at the point when a desired amount of reduced coenzyme Q10 is produced. The culture duration is not particularly limited and it is generally 20 to 200 hours. The above-mentioned culture is generally carried out aerobically. The term "aerobically" means a condition that oxygen is supplied so as not to cause oxygen limitation (oxygen deficiency) during the culture, and preferably a condition that oxygen is supplied sufficiently so as not to cause substantial oxygen limitation during the culture. The culture is carried out generally under an aeration condition, preferably under an aeration and stirring condition. By using the above-mentioned microorganisms and culture conditions, it becomes possible to obtain microbial cells containing reduced coenzyme Q10 at a ratio of not less than 70 mole %, preferably not less than 75 mole % among the entire coenzymes Q10. Furthermore, the productivity of reduced coenzyme Q10 of as high as not less than 1 g/mL, preferably not less than 2 (g/mL, and still more preferably not less than 3 \|xg/mL can be obtained. Next, recovery of the reduced coenzyme Q10 produced by the above-mentioned culture will be described. In the present invention, an efficient production of reduced coenzyme Q10 on the industrial scale is made to be possible partially by the above-mentioned suitable culture and partially by the suitable recovery process of reduced coenzyme Q10 as described below. Recovery of reduced coenzyme Q10 is carried out by extraction from the microbial cells obtained by the above- mentioned culture using an organic solvent. In the extraction, cells can be disrupted optionally. The cell disruption contributes to the efficient extraction of the reduced coenzyme Q10 produced and accumulated in cells. It is needless to say that the cell disruption and extraction can be carried out at the same time. Incidentally, "disruption" in the present invention may be carried out to the extent that the surface structure such as a cell wall is broken so as to make extraction of reduced coenzyme Q10 possible; therefore, it is not necessary that microbial cells are torn or fragmentated. The above-mentioned cell disruption is not necessarily required in the case of bacteria. However, in the case of yeast or fungi, the cell disruption is generally required and, when cells are not disrupted, it becomes difficult to efficiently recover the reduced coenzyme Q10 produced and accumulated in the cells. The above-mentioned disruption of microbial cells can De carried out by the following one or several disruption nethods in optional order. As the disruption method, there nay be mentioned, for example, a physical treatment, a chemical treatment., an enzymic treatment as well as a heating treatment, an autolysis, an osmolysis, a plasmoptysis and the like. The above-mentioned physical treatment can be carried out, for example, by using a high pressure homogenizer, an ultrasonic homogenizer, a French press, a ball mill and the like or using them in combination. The above-mentioned chemical treatment can be carried out, for example, by using an acid (preferably a strong acid) such as hydrochloric acid and sulfuric acid, a base (preferably a strong base) such as sodium hydroxide and potassium hydroxide and the like or using them in combination. The above-mentioned enzymic treatment can be carried out, for example, by using lysozyme, zymolyase, glucanase, Novozyme, protease, cellulase and the like or by using them appropriately in combination. The dbove-mentioned heating treatment can be carried out, for example, by heating to the temperature range of 60 to 100°C for about 30 minutes to 3 hours. The above-mentioned autolysis can be carried out, for example, by treatment with a solvent such as ethyl acetate. The osmolysis or the plasmoptysis for disrupting cells by treating cells with a solution having a different salt concentration from that in the cells are often combinedly used with the above-mentioned physical treatment, chemical treatment, enzymic treatment, heating treatment, autolysis and/or the like since the above lytic method alone is insufficient in the disruption effect. As the cell disruption method as a pretreatment of extraction and recovery of reduced coenzyme Q10, among the above-mentioned disruption methods, the physical treatment, the chemical treatment (particularly, an acid treatment and preferably the one with a strong acid (e.g. an acid having a pKa value of not more than 2.5 in the form of an aqueous solution) under the condition that reduced coenzyme Q10 is protected from an oxidation reaction as described below) and the heating treatment are preferred. From the viewpoint of disruption efficiency, the physical treatment is more preferred. A conventional cell disruption method and coenzyme Q10 extraction method, specifically, a method comprising extracting coenzyme Q10 by an organic solvent in the presence of sodium hydroxide and pyrogallol has problems in terms of cost, waste treatment, safety in effective utilization of waste microorganisms (waste cells) such as recovery of protein, and the like. However, the cell disruption method, particularly the physical treatment method of the present invention, does not cause subgeneration of a large quantity of salts by neutralization, and is a suitable method from a viewpoint of the waste treatment and the effective utilization of waste microorganisms (wasle cells). The form of the microbial cells to be used for the above-mentioned cell disruption may be a broth, a concentrated broth, microbial cells collected as wet cells from the broth, a product obtained by washing them, a suspension of the wet cells in a solvent (including, for example, water, physiological saline solution, buffers and the like), dry cells obtained by drying the above-mentioned wet cells, a suspension of the dry cells in a solvent (including, for example, water, physiological saline solution, buffers and the like), and the like. Preferred is an aqueous suspension of microbial cells, and in terms of operability and the like, more preferred are the broth, the concentrated broth, and the product obtained by washing them. The form of the above-mentioned microbial cells or disrupted product thereof to be used for extraction and recovery of reduced coenzyme Q10 is, similarly as described above, not particularly limited and may be wet cells/dry cells of the microbial cells/disrupted product thereof. Preferably, it is an aqueous suspension of the microbial cells or disrupted product thereof, and more preferably the broth, the concentrated and/or washed broth, or solutions obtained by disrupting them (each of them is an aqueous suspension). The cell concentration in the above-mentioned suspension of the microbial cells or disrupted product thereof is not particularly limited and is generally 1 to 25% by weight on the basis of dry weight. Preferably, it is 10 to 20% by weight in terms of cost. Reduced coenzyme Q10 can be recovered by extracting the microbial cells and disrupted product thereof obtained in such a manner by an organic solvent. As the organic solvent to be used for the extraction, there may be mentioned hydrocarbons, fatty acid esters, ethers, alcohols, fatty acids, ketones, nitrogen compounds (including nitriles and amides), sulfur compounds and the like. Particularly, in extracting reduced coenzyme Q10, in terms of protection from oxidation by a molecular oxygen, at least one species of hydrocarbons, fatty acid esters, ethers, and nitriles is preferably used. Among them, hydrocarbons and fatty acid esters are particularly preferable, and hydrocarbons are most preferable. On the industrial production scale, complete oxygen elimination is very difficult to be achieved and, furthermore, fairly long periods of time are required for individual operations, unlike laboratory scale production, so that residual oxygen exerts a great adverse effect. The oxidation in question is directly connected to a subgeneration of oxidized coenzyme Q10 from reduced coenzyme Q10. Accordingly, use of the above-mentioned organic solvent (such as hydrocarbons, fatty acid esters, ethers, and nitriles) with high oxidation prevention effect in the extraction of reduced coenzyme Q10 assists an efficient extraction. The hydrocarbons are not particularly restricted, but there may be mentioned, for example, aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, and the like. Preferred are aliphatic hydrocarbons and aromatic hydrocarbons, and more preferred are aliphatic hydrocarbons. The aliphatic hydrocarbons are not particularly restricted, and may be cyclic or acyclic, or saturated or unsaturated. However, generally, saturated ones are preferably used. Usually, ones containing 3 to 20 carbon atoms, preferably 5 to 12 carbon atoms, and more preferably 5 to 8 carbon atoms are used. As specific examples, there may be mentioned, for example, propane, butane, isobutane, pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2- dimethylbutane, 2,3-dimethylbutane, heptane, heptane isomers (e.g. 2-methylhexane, 3-methylhexane, 2,3- dimethylpentane, 2,4-dimethylpentane), octane, 2,2,3- trimethylpentane, isooctane, nonane, 2,2,5-trimethylhexane, decane, dodecane, 2-pentene, 1-hexene, 1-heptene, 1-octene, 1-nonene, 1-decene, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, p- menthane, cyclohexene, and the like. Preferred are pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane, 2,3-dimethylbutane, heptane, heptane isomers (e.g. 2- methylhexane, 3-methylhexane, 2,3-dimethylpentane, 2,4- dimethylpentane), octane, 2, 2, 3-trimethylpentane, isooctane, nonane, 2,2,5-trimethylhexane, decane, dodecane, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, p-menthane, and the like. More preferred are pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane, 2, 3-dimethylbutane, heptane, heptane isomers (e.g. 2-methylhexane, 3- methylhexane, 2,3-dimethylpentane, 2,4-dimethylpentane), octane, 2,2,3-trimethylpentane, isooctane, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, and the like. Generally, heptanes, not only heptane but also heptane isomers such as methylcyclohexane having 7 carbon atoms and a mixture thereof are preferably used. More preferred are pentanes (e.g. pentane and the like) having 5 carbon atoms, hexanes (e.g. hexane, cyclohexane and the like) having 6 carbon atoms, and heptanes (e.g. heptane, mRthylcyclohexane and the like) having 7 carbon atoms. Particularly preferred are heptanes (e.g. heptane, methylcyclohexane and the like) in terms of especially high protection effect from oxidation, and the most preferred is heptane. The aromatic hydrocarbons are not particularly restricted, but generally ones containing 6 to 20 carbon atoms, preferably 6 to 12 carbon atoms, and more preferably 7 to 10 carbon atoms are used. As specific examples, there may be mentioned, for example, benzene, toluene, xylene, o- xylene, m-xylene, p-xylene, ethylbenzene, cumene, mesitylene, tetralin, butylbenzene, p-cymene, cyclohexylbenzene, diethylbenzene, pentylbenzene, dipentylbenzene, dodecylbenzene, styrene, and the like. Preferred are toluene, xylene, o-xyiene, m-xylene, p-xylene, ethylbenzene, cumene, mesitylene, tetralin, butylbenzene, p-cymene, cyclohexylbenzene, diethylbenzene, pentylbenzene and the like. More preferred are toluene, xylene, o-xylene, m-xylene, p-xylene, cumene, tetralin and the like, and most preferred is cumene. The halogenated hydrocarbons are not particularly restricted, and may be cyclic or acyclic, or saturated or unsaturated. However, acyclic ones are preferably used in general. Usually, more preferred are chlorinated hydrocarbons and fluorinated hydrocarbons, and chlorinated hydrocarbons are still more preferred. Additionally, ones containing 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and more preferably 1 to 2 carbon atoms are suitably used. As specific examples, for example, there may be mentioned dichloromethane, chloroform, carbon tetrachloride, 1, 1-dichloroethane, 1,2-dichloroethane, 1,1,1- trichloroethane, 1,1.2-trichloroethane, 1,1,1,2- tetrachloroethane,- 1, 1, 2, 2-tetrachloroethane, pentachloroethane, hexachloroethane, 1,1-dichloroethylene, 1, 2-dichloroethylene, trichloroethylene, tetrachloroethylene. 1.2-dichloropropane, 1,2,3- trichloropropane, chlorobenzene, 1,1,1,2-tetrafluoroethane, and the like. Preferred are dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2- dichloroethane, 1,1,1-trichloroethane, 1,1,2- trichloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene, chlorobenzene, 1,1,1,2-tetrafluoroethane, and the like. More preferred are dichloromethane, chloroform, 1,2-dichloroethylene, trichloroethylene, chlorobenzene, 1,1,1,2-tetrafluoroethane and the like. The fatty acid esters are not particularly restricted, but there may be mentioned, for example, propionates, acetates, formates, and the like. Preferred are acetates and formates, and more preferred are acetates. Ester functional groups thereof are not particularly restricted, but, in general, preferred are alkyl esters having 1 to 8 carbon atoms and aralkyl esters having 7 to 12 carbon atoms, more preferred are alkyl esters having 1 to 6 carbon atoms, and still more preferred are alkyl esters having 1 to 4 carbon atoms. As specific examples of the propionates, there may be mentioned, for example, methyl propionate, ethyl propionate, butyl propionate, isopentyl propionate, and the like. Preferred are ethyl propionate and the like. As specific examples of the acetates, there may be mentioned, for example, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, sec-butyl acetate, pentyl acetate, isopentyl acetate, sec-hexyl acetate, cyclohexyl acetate, benzyl acetate, and the like. Preferred are methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, sec-butyl acetate,- pentyl acetate, isopentyl acetate, sec-hexyl acetate, cyclohexyl acetate, and the like. More preferred are methyl acetate, ethyl acetate, prcpyl acetate, isopropyl acetate, butyl acetate, acetate. As specific examples of the formates, there may be mentioned, for example, methyl formate, ethyl formate, propyl formate, isopropyl formate, butyl formate, isobutyl formate, sec-butyl formate, pentyl formate, and the like. Preferred are methyl formate, ethyl formate, propyl formate, butyl formate, isobutyl formate, pentyl formate, and the like. Most preferred is ethyl formate. The ethers are not particularly restricted, and may be cyclic or acyclic, or saturated or unsaturated. But saturated ones are preferably used in general. Generally, ones containing 3 to 20 carbon atoms, preferably 4 to 12 carbon atoms and more preferably 4 to 8 carbon atoms are used. As specific examples, there may be mentioned, for example, diethyl ether, methyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, dihexyl ether, ethyl vinyl ether, butyl vinyl ether, anisol, phenetole, butyl phenyl ether, methoxytoluene, dioxane, furan, 2- methylfuran, tetrahydrofuran, tetrahydropyran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, ethylene glycol dibutyl ether, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, and the like. Preferred are diethyl ether, methyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, dihexyl ether, anisol, phenetole, butyl phenyl ether, methoxytoluene, dioxane, 2-methylfuran, tetrahydrofuran, tetrahydropyran, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, ethylene glycol dibutyl ether, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, and the like. More preferred are diethyl ether, methyl tert-butyl ether, anisol,- dioxane, tetrahydrofuran, ethylene glycol. monomethyl ether, ethylene glycol monoethyl ether, and the like. Still more preferred are dietbvl ether- inethvl tert-butv] ether- anisol - and the like, and most preferred is methyl tcrt-butyl ether. The alcohols are not particularly restricted but may be cyclic or acyclic, or saturated or unsaturated. Saturated ones are generally preferred, however. Generally, ones containing 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms, and still more preferably 1 to 6 carbon atoms are used. Among them, monohydric alcohols containing 1 to 5 carbon atoms, dihydric alcohols containing 2 to 5 carbon atoms, and trihydric alcohols containing 3 carbon atoms are preferred. As specific examples of these alcohols, there may be mentioned, for example, monohydric alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2- butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-l-butanol.. isopentyl alcohol, tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol, 1-hexanol, 2-methyl-l-pentanol, 4-methyl-2- pentanol, 2-ethyl-l-butanol, 1-heptanol, 2-heptanol, 3- heptanol, 1-octanol, 2-octanol, 2-ethyl-l-hexanol, 1- nonanol, 1-decanol, 1-undecanol, 1-dodecanol, allyl alcohol, propargyl alcohol, benzyl alcohol, cyclohexanol, 1- methylcyclohexanol, 2-methylcyclohexanol, 3- methylcyclohexanol, 4-methylcyclohexanol, and the like; dihydric alcohols such as 1,2-ethanediol, 1,2-propandiol, 1,3-propandiol, 1,2-butanediol, 1,3-butanediol, 1,4- butanediol, 2,3-butanediol, 1,5-pentanediol, and the like; and trihydric alcohols such as glycerol, and the like. As the monohydric alcohols, preferred are methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 2- pentanol, 3-pentanol, 2-methyl-l-butanol, isopentyl alcohol, tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol, 1-hexanol, 2-methyl-l-pentanol, 4-methyl-2-pentanol, 2- ethyl-1-butanol, 1-heptanol, 2-heptanol, 3-heptanol, 1- octanol, 2-octanol, 2-ethyl-l-hexanol, 1-nonanol, 1-decanol, 1-undecanol, 1-dodecanol, benzyl alcohol, cyclohexanol, 1- methylcyclohexanol, 2-methylcyclohexanol, 3- methylcyclohexanol, 4-methylcyclohexanol, and the like. More preferred are methanol, ethanol, 1-propanol, 2- propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert- butyl alcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2- methyl-1-butanol, isopentyl alcohol, tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol, 1-hexanol, 2-methyl- i-pentanol, 4-methyi-2-pentanoi, 2-ethyi-l-butanoi, cyclohexanoi, and the like. Still more preferred are methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2- butanoi, isobutyi alcohol, tert-butyl alcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-l-butanol, isopentyl alcohol, tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol, and the like. Particularly preferred are methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, 2-methyl-l-butanol, isopentyl alcohol, and the like. Most preferred is 2-propanol. As the dihydric alcohols, preferred are 1,2- ethanediol, 1,2-propandiol, 1, 3-propandiol, and the like. Most preferred is 1,2-ethanediol. As the trihydric alcohols, glycerol is preferred. As fatty acids, there may be mentioned, for example, formic acid, acetic acid, propionic acid, and the like. Preferred are formic acid and acetic acid, and most preferred is acetic acid. The ketones are not particularly restricted, and ones having 3 to 6 carbon atoms are preferably used. As specific examples, there may be mentioned, fo'r example, acetone, methyl ethyl ketone, methyl butyl ketone, methyl isobutyl ketone, and the like. Preferred are acetone and methyl ethyl ketone, and most preferred is acetone. The nitriles are not particularly restricted, and may be cyclic or acyclic, or saturated or unsaturated. However, saturated ones are preferably used in general. Generally, ones containing 2 to 20 carbon atoms, preferably 2 to 12 carbon atoms, and more preferably 2 to 8 carbon atoms are used. As specific examples, there may be mentioned, for example, acetonitrile, propiononitrile, malononitrile, butyronitrile, isobutyronitrile, succinonitrile, valeronitrile, glutaronitrile, hexanenitrile, heptylcyanide, octylcyanide, undecanenitrile, dodecanenitrile, tridecanemtrile, pentadecanenitriie, stearonitriie, chloroacetonitriie, bromoacetonitrile, chloropropiononitrile, bromopropiononitrile, methoxyacetonitriie, methyl cyanoacetate, ethyl cyanoacetate, tolunitrile, benzonitrile, chlorobenzonitrile, bromobenzonitrile, cyanobenzoic acid, nitrobenzonitrile, anisonitrile, phthalonitrile, bromotolunitrile, methyl cyanobenzoate, methoxybenzonitrile, acetylbenzonitrile, naphthonitrile, biphenylcarbonitrile, phenylpropiononitrile, phenylbutyronitrile, methylphenylacetonitrile, diphenylacetonitrile, naphthylacetonitrile, nitrophenylacetonitrile, chlorobenzylcyanide, cyclopropanecarbonitrile, cyclohexanecarbonitrile, cycloheptanecarbonitrile, phenylcyclohexanecarbonitrile, tolylcyclohexanecarbonitrile, and the like. Preferred are acetonitrile, propiononitrile, succinonitrile, butyronitrile, isobutyronitrile, valeronitrile, methyl cyanoacetatev ethyl cyanoacetate, benzonitrile, tolunitrile and chloropropiononitrile. More preferred are acetonitrile, propiononitrile, butyronitrile and isobutyronitrile, and most preferred is acetonitrile. As the nitrogen compounds other than nitriles, there may be mentioned, for example, amides such as formamide, N- methylformamide, N, N-dimethylformamide, N,N- dimethylacetoamide, N-methylpyrrolidone, and nitromethane, triethylamine, pyridine, and the like. As the sulfur compounds, there may be mentioned, for example, dimethyl sulfoxide, sulfolane, and the like. In selecting the organic solvent to be used from among the organic solvents mentioned above, such properties as boiling point and viscosity (e.g. the solvent should have a boiling point which allows appropriate warming for increasing solubility and facilitates a solvent removal from wet masses by drying and solvent recovery from crystallization filtrates and the like (about 30 to 150°C at 1 atm), a melting point such that solidification hardly occurs in handling at room temperature as well as upon cooling to room temperature or below (not lower than about 0"C, preferably not lower than about 1U°C, more preferably not lower than about 20°C), and a low viscosity (not higher than about 10 cp at 20°C and the like)) are preferably taken into consideration. The oxidation prevention effect on reduced coenzyme Q10 in a solvent tends to increase in a highly-concentrated solution of reduced coenzyme Gh0. Reduced coenzyme Q10 shows high solubility in the above-mentioned organic solvents with high oxidation prevention effect (e.g. hydrocarbons, fatty acid esters and the like). The high solubility makes it possible to handle the highly- concentrated solution and to promote the oxidation prevention. A preferable concentration of reduced coenzyme Q10.for oxidation prevention at the time of extraction is not particularly limited, but is generally not less than 0.001% by weight, preferably not less than 0.01% by weight, and more preferably not less than 0.1% by weight as the concentration of reduced coenzyme Q10 in the above- mentioned organic solvent. The upper limit is not particularly limited, however, in general, it is not more than 10% by weight. Among the above-mentioned organic solvents, to extract and recover reduced coenzyme Q10 from wet cells and dry cells of the microbial cells or disrupted product thereof, hydrophilic organic solvents are preferably used. Specifically, there may be mentioned acetone, acetonitrile, methanol, ethanol, 1-propanol, 2-propanol and the like. Furthermore, among the above-mentioned organic solvents, to extract and recover reduced coenzyme Q10 from the aqueous suspension of the microbial cells or disrupted product thereof, hydrophobic organic solvents are preferably used. Use of such solvents assists the removal ot water-soluble substances derived from microorganisms. Many of hydrophobic organic solvents have high oxidation prevention effect as described above, thus are very advantageous. As the hydrophobic organic solvents, hydrocarbons, fatty acid esters and ethers are preferred. In the case of the above-mentioned extraction operation, when reduced coenzyme Q10 is extracted from the aqueous suspension of the microbial cells or disrupted product thereof, particularly from the aqueous suspension of the disrupted product, further particularly the case in which the disrupted product is physically treated, by an organic solvent, emulsions tend to be partly formed because of the presence of cell components such as proteins and phase separation tends to be difficult. Therefore, it becomes important to suppress the formation of emulsions mentioned above and to efficiently carry out extraction. For that, as an extraction solvent, in addition to the above-mentioned hydrophobic organic solvent, it is preferable to use a hydrophilic organic solvent as an auxiliary solvent in combination. In this case, the hydrophobic organic solvent is not particularly limited and those mentioned above may be used. Preferred are hydrocarbons, and more preferred are aliphatic hydrocarbons. Among the aliphatic hydrocarbons, those having 5 to 8 carbon atoms are preferably used. As specific examples of the aliphatic hydrocarbons containing 5 to 8 carbon atoms, there may be mentioned, for example, pentane, 2-methylbutane, hexane, 2-methylpentane, 2, 2-dimethylbutane, 2,3-dimethylbutane, heptane, heptane isomers (e.g. 2-methylhexane, 3-methylhexane, 2,3- dimethylpentane, 2,4-dimethylpentane), octane, 2,2,3- trimethylpentane, isooctane, cyclopentane, methylcyclopentane, cyclohexane, methylcyclohexane, ethylcyclohexane, and the like. Particularly preferred are hexane, heptane and methylcyclohexane, and most preferred are hexane and heptane. The hydrophilic organic solvent to be used in combination with the above-mentioned hydrophobic organic solvent is not particularly limited and those mentioned above may be used. Preferred are alcohols. Among the alcohols, monohydric alcohols having 1 to 5 carbon atoms are preferably used. As specific examples thereof, there may be mentioned, for example, methanol, ethanol, 1- propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 2-pentanol, 3- pentanol, 2-methyl-l-butanol, isopentyl alcohol, tert- pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol, and the like. Particularly preferred are methanol, ethanol, 1- propanol and 2-propanol, and most preferred is 2-propanol. The amounts of the above-mentioned hydrophilic organic solvent and hydrophobic organic solvent to be used are not particularly limited. But preferably, as the concentration at the time of extraction, the hydrophilic organic solvent is used in a range of 5 to 50% by volume and the hydrophobic organic solvent is used in a range of 25 to 65% by volume relative to the total volume of the entire solution. In recovering reduced coenzyme Q10, the temperature at the time of extraction is not particularly limited and is generally in a range of 0 to 60°C and preferably 20 to 50°C. As the extraction method, both batch extraction and continuous extraction (preferably countercurrent multistage extraction) may be used. However, the continuous extraction (preferably countercurrent multistage extraction) is preferable in terms of productivity. The stirring duration in the batch extraction is not particularly limited but is generally not less than 5 minutes. The average retention time in the continuous extraction is not particularly limited but is generally nut less than 10 minutes. In recovering reduced coenzyme Q10, it is preferable to be careful so that reduced coenzyme Q10 is not decomposed (e.g. so that reduced coenzyme Q10 is not oxidized to oxidized coenzyme Q10) For that, the above- mentioned extraction (including cell disruption) is preferably carried out under an acidic to a weakly basic condition, and more preferably under an acidic to a neutral condition. In the case where a pH is used as an index, although it depends on the contact time, the pH is generally not more than 10, preferably not more than 9, more preferably not more than 8, and still more preferably not more than 7. By the above-mentioned conditions, an oxidation reaction can be substantially prevented and, optionally, more strictly, the above-mentioned cell disruption and/or extraction are preferably carried out under the condition that reduced coenzyme Q10 is protected from an oxidation reaction. It is preferable to carry out at least the extraction under this condition, and it is more preferable to carry out the disruption and the extraction under this condition. As "the condition that reduced coenzyme Q10 is protected from an oxidation reaction" means, for example, a deoxygenized atmosphere (an atmosphere of an inert gas such as nitrogen gas, carbon dioxide gas, helium gas, argon gas or hydrogen gas, reduced pressure, a boiling condition); a high salt concentration condition, for example, preferably a condition where salts (e.g. inorganic salts such as sodium chloride and sodium sulfate) are contained in not less than about 5% in an aqueous phase; the condition in the presence of a strong acid (e.g. an acid with a pKa value of not more than 2.5 in an aqueous solution), for example, in the presence of not less than 0.1 mole % of the strong acid relative to i mole of reduced coenzyme Q10; and the condition in the presence of an antioxidant, for example, in the concomitant presence of ascorbic acid, citric acid, salts and esters thereof (e.g. not less than 0.1% by weight of them relative to reduced coenzyme Q10) . There may also be mentioned a reduction condition (a condition in which oxidized coenzyme Q10 can be converted into reduced coenzyme Q10) , for example, a condition involving a contact with a reducing agent such as dithionous acid. By the above-mentioned culture (fermentation) and extraction, reduced coenzyme Q10 can be suitably produced and recovered. Preferably, an extract containing not less than 70 mole %, preferably not less than 75 mole % of reduced coenzyme Q10 among the entire coenzymes Q10 is obtained. Thus-obtained extract containing reduced coenzyme Q10 is optionally purified by column chromatography, reduction treatment, or the like and then subjected to crystallization to obtain high-purity reduced coenzyme Q10 crystals. Incidentally,- also in this case, a' series of treatment steps are preferably carried out under "the condition that reduced coenzyme Q10 is protected from an oxidation reaction" mentioned above. In the present invention, oxidized coenzyme Q10 can be produced by oxidizing the above-mentioned microbial cells or disrupted product thereof and then extracting oxidized coenzyme Q10 by an organic solvent, or extracting reduced coenzyme Q10 from the microbial cells or disrupted product thereof by an organic solvent, purifying optionally and oxidizing the resultant to oxidized coenzyme Q10. The above-mentioned oxidation may be carried out by, for example, mixing reduced coenzyme Q10 (preferably an aqueous suspension of the microbial cells or disrupted product thereof containing reduced coenzyme Q10, an extract containing reduced coenzyme Q10 or the like) with an oxidizing agent (e.g. manganese dioxide or the like) and then, for example, oxidizing the mixture at room temperature (e.g. 30°C) for not less than 30 minutes.. In the case where the microbial cells or disrupted product thereof are oxidized, the extraction operation of oxidized coenzyme Q10 can be carried out in the same manner as the above-mentioned extraction operation of reduced coenzyme Q10. Thereby, oxidized coenzyme Q10 can be efficiently recovered. Incidentally, it is not necessary to carry out the recovery of oxidized coenzyme Q10 under "the condition that reduced coenzyme Q10 is protected from an oxidation reaction", which is recommended for the recovery of reduced coenzyme Q10 and the recovery may be carried out in consideration of general safe operation and the like. The thus-obtained oxidized coenzyme Q10 may be optionally purified by column chromatography or the like, and, finally by conducting crystallization operation, high-purity oxidized coenzyme Q10 crystals may be obtained. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a schematic diagram of a countercurrent 3-step continuous extraction apparatus used in Example 8. BEST MODE FOR CARRYING OUT THE INVENTION The following examples illustrate the present invention in further detail. These examples are, however, by no means limitative of the scope of the present invention. (Example 1) Various coenzyme Q10-producing microorganisms shown in the following Tables 1 to 3 were cultured with shaking (amplitude: 2 cm, 310 reciprocation/min) at 25°C for 72 hours in 10 mL of culture media [(glucose: 20 g, peptone: 5 g, yeast extract: 3 g, malt extract: 3 g)/L, pH: 6.0] using test tubes (inner diameter: 21 mm, entire length: 200 mm), and the obtained broth were optionally concentrated. Under a nitrogen atmosphere, in the concomitant presence of 3 parts by volume of isopropanol and 18.5 parts by volume of n-hexane relative to 10 parts by volume of the broth, the obtained solutions were vigorously shaken for 3 minutes using 10 parts by volume of glass beads (425 to 600 \|im) to carry out cell disruption and extraction. The obtained hexane phases were evaporated (at 40°C) under reduced pressure and analyzed by high performance liquid chromatography (HPLC) to determine the ratio and the production amount of reduced coenzyme Q10. HPLC conditions Column: YMC-Pack 4.6 x 250 mm (manufactured by YMC. Co., Ltd.) Mobile phase: methanol/n-hexane = 85/15 Flow rate: 1 mL/min Detection: UV 275 ran The results are shown in Tables 1 to 3. The ratio of reduced coenzyme Q10 means a mole percentage value of the ratio of reduced coenzyme Q10 relative to the total of oxidized coenzyme Q10 and reduced coenzyme Q10 on the basis of the areas of the peaks of reduced coenzyme Cho and oxidized coenzyme Cho and the ratio of the mole absorption coefficients thereof (1 : 7.5). 38 (Example 2) Rhodotorula glutinis IFO1125 was aerobically cultured at 25°C for 48 hours in a culture medium (peptone: 5 g, yeast extract: 3 g, malt extract: 3 g, glucose: 20 g/L, pH: 6.0) . The cells after the culture were collected by centrifugation and suspended in a phosphoric acid buffer solution at pH 7 to which N-methyl-N'-nitro-N- nitrosoguanidine have been added so as to have its concentration of 200 g/mL. After maintaining the solution at 2 5°C for 1 hour, the cells were washed for 5 times with a 0.9% NaCl solution and further suspended in a 0.9% NaCl solution. The obtained cell suspension was properly diluted and a colony was to be formed on an agar plate of the above-mentioned culture medium. The production amount and the ratio of reduced coenzyme Q10 in the isolated mutant strain were determined in the same manner as Example 1. The strains having higher production amount and the ratio of reduced coenzyme Q10 as compared with those of wild strains was further mutated repeatedly. As the result, by repeating the mutagenesis for 10 times, mutant strains with productivity of not less than 15 g/mL were obtained. In this case, the ratio of reduced coenzyme Qlo was not less than 80 mole %. (Example 3) Saitoella complicata IFO 10748 was aerobically cultured at 25°C for 72 hours in 10 L of a culture medium (peptone: 5 g, yeast extract: 3 g, malt extract: 3 g, glucose: 20 g/L, pH: 6.0). The obtained cells were disrupted for 2 times at 80 MPa of disruption pressure by a pressure homogenizer (manufactured by Lanni Co.) sealed with nitrogen gas to obtain a cell-disrupted solution. The cell-disrupted solution was subjected to extraction with 30 parts by volume of isopropanol and 40 parts by volume of hexane for 3 times to obtain an extract. The extraction ratio was 99%. The ratio of reduced coenzyme Q10 was 97 mole %. (Example 4) When mutant strains of Rhodotorula glutinis IFO1125 were aerobically cultured at 25°C in 10 L of a culture medium (peptone: 10 g, yeast extract: 5 g, malt extract: 3 g, glucose: 20 g/L, pH: 6.0), glucose was fed at the rate of 4 g/h after the lapse of 48 hours to 96 hours (fed glucose amount: 190 g). The production amount of reduced coenzyme Q10 per culture medium was not less than 2 0 ng/mL and the ratio of reduced coenzyme Q10 was not less than 80 mole %. (Example 5) The extract obtained in Example 3 was subjected to solvent substitution with a hexane solution, the resultant was adsorbed in a column filled with silica gel and subjected to development and elution by a solution of n- hexane/diethyl ether (9/1) to obtain a fraction containing reduced coenzyme Q10. Furthermore, the fraction was cooled to 2°C with stirring to obtain a white slurry. All the above-mentioned operations were carried out in a nitrogen atmosphere. The obtained slurry was filtered under reduced pressure, the resulting wet crystals were washed with the development solution same as used above (the temperature of the solvent used for washing was 2°C) , and the wet crystals were dried under reduced pressure (20 to 40°C, 1 to 30 mmHg) to obtain 81 mg of white dry crystals. The purity of the obtained crystals was 99.9% and the ratio of reduced coenzyme Q10 was 90 mole %. (Example 6) The extract obtained in Example 3 was subjected to solvent substitution with n-hexane, the resultant was added with 50 mg of manganese dioxide, and the mixture was stirred at 30°C for 30 minutes. Thus-obtained reaction solution was fractionated and purified in the same manner as Example 5 to obtain 74 mg of high-purity oxidized coenzyme Q10. (Example 7) Saitoella complicata IFO 10748 was aerobically cultured at 25°C for 72 hours in 500 mL of a culture medium (peptone: 5 g, yeast extract: 3 g, malt extract: 3 g, glucose: 20 g/L, pH: 6.0) . The obtained cells were disrupted for 2 times at 80 MPa of disruption pressure by a pressure homogenizer (manufactured by Lanni Co.) sealed with nitrogen gas to obtain a cell-disrupted solution. The ratio of reduced coenzyme Q10 in the cell-disrupted solution was 97% relative to the entire coenzymes Q10 including oxidized coenzyme Q10. 200 mL of the cell- disrupted solution was mixed with isopropanol and n-hexane at the ratios shown in the first extraction section in the following Table 4 so as to adjust the total solvent amount to be 500 mL and the mixtures were stirred at 40°C for 30 minutes to carry out the tirst extraction. After completion of the extraction, the resultants were kept standing for 10 minutes and the separated upper layers were collected. The volume ratios of the lower layers (residues) relative to the total solution amounts were defined as indexes of separability and shown as the interface positions in Table 4. Furthermore, in order to carry out the second extraction, the solvent concentrations of the residual layers were measured and isopropanol and hexane were further added so as to keep the solvent ratios in the entire solutions be the ratios shown in the second extraction section in Table 4. The resulting solutions were stirred at 40°C for 30 minutes. Then, the solutions were kept standing for 10 minutes and the upper layers were collected in the same manner as described above to determine the solvent concentrations of the residual layers Isopropanol and hexane were added thereto so as to keep the solvent ratios in the entire solutions be the ratios shown in the third extraction section in Table 4, and the solutions were stirred at 25°C for 30 minutes to carry out the third extraction. The ratios of the amounts of reduced coenzyme Q10 contained in the collected upper layers of each of the first, second and third steps relative to the amount of reduced coenzyme Q10 contained in the cell-disrupted solution or the extraction residue before the extraction were defined as the extraction ratios of reduced coenzyme Q10 in the respective steps. The calculation results are shown in Table 4. The integrated extraction ratios of reduced coenzyme Q10 in the second and third extraction steps are also shown. In any steps, the static separability was excellent and the integrated extraction ratio in the case where extraction was repeated for 3 times was as high as not less than 90% to show high recovery ratio. Particularly, in the case where the isopropanol concentration was adjusted to be not less than 30%, the recovery ratio was as high as not less than 99%. (Example 8) Saitoella complicata IFO 10748 was aerobically cultured at 25UC for 72 hours in 7bO L of a culture medium (peptone: 5 g, yeast extract: 3 g, malt extract: 3 g, glucose: 20 g/L, pH: 6.0). The obtained cells were disrupted for 2 times at 140 MPa of disruption pressure by a pressure homogenizer (manufactured by Lanni Co.) sealed with nitrogen gas to obtain a cell-disrupted solution. The cell-disrupted solution was subjected to continuous extraction by a countercurrent 3-step continuous extractioi apparatus shown in Fig. 1. The capacity of the stirring tank was 630 L and the capacity of the static separation tank was 200 L. The cell-disrupted solution was supplied to the first stirring tank and isopropanol and n-hexane were supplied to respective steps. The supply amount of the cell-disrupted solution was 2 L/min and the supply amounts of isopropanol and n-hexane were adjusted to be 1.3 L/min for isopropanol and 3.7 L/min for n-hexane as the total of the supply amounts in respective steps. In this case, the solvent concentration in respective steps was properly adjusted so that the isopropanol concentration of 5 to 50 v/v % and the n-hexane concentration of 25 to 65 v/v % were kept. The extraction temperature was 40°C and the treatment duration was 6 hours. At the point after the lapse of 6 hours, the recovery ratio of reduced coenzyme Q10 extracted from the cell-disrupted solution was calculated on the basis of reduced coenzyme Q10 remaining in the extraction residue in the static separation tank in the third step to find the recovery ratio of 98.9%. The static separation was well carried out during the entire operation period and stable continuous extraction was possible. INDUSTRIAL APPLICABILITY According to the processes of the present invention, reduced coenzyme Q10 can be produced cheaply on the industrial scale by considerably simple steps comprising cuituring microorganisms and recovering reduced coenzyme Q10. In addition, oxidized coenzyme Q10 can also be produced by simple processes. WE CLAIM: 1. A process for producing the oxidized coenzyme Q10 represented by the following formula (I): which comprises culturing reduced coenzyme Q10 producing microorganisms in a culture medium containing a carbon source, a nitrogen source, a phosphorus source and a micronutrient to obtain microbial cells containing reduced coenzyme Q10 at a ratio of not less than 70 mole % among the entire coenzymes Q10 optionally disrupting the microbial cells; and either oxidizing thus-produced reduced coenzyme Q10 to oxidized coenzyme Q10 using an oxidizing agent and then extracting the resultant by an organic solvent, or extracting thus-producing reduced coenzyme Q10 by an organic solvent, purifying optionally and oxidizing the resultant to oxidized coenzyme Q10 using an oxidizing agent. 2. The process as claimed in Claim 1, wherein the production amount of reduced coenzyme Q10 on completion of the culture is not less than 1 g/mL, 3. The process as claimed in Claim 1 or 2, wherein the culture is carried out at 15 to 45°C and at a pH of 4 to 9. 4. The process as claimed in any one of Claims 1 to 3, wherein the concentration of the carbon source in the culture is controlled to a concentration that no substantially adverse effects are caused on the productivity of reduced coenzyme Q10. 5. The process as claimed in Claim 4, wherein the culture is carried out by a fed batch culture method. 6. The process as claimed in Claim 5, wherein the carbon source is supplied to the culture medium separately from other components. 7. The process as claimed in any one of Claims 1 to 6, wherein the culture is carried out aerobically. 8. The process as claimed in any one of Claims 1 to 7, wherein the microbial cells are disrupted in the extraction. 9. The process as claimed in Claim 8. wherein the cell disruption is carried out by a physical treatment, a chemical treatment, an enzymic treatment, a heating treatment, an autolysis, an osmolysis or a plasmoptysis. 10. The process as claimed in Claim 9, wherein the cell disruption is carried out by a physical treatment. 11. The process as claimed in Claim 10, wherein the physical treatment is carried out by a high pressure homogenizer, an ultrasonic homogenizer, a French press or a ball mill. 12. The process as claimed in any one of Claims 1 to 11, wherein the extraction of coenzymes Q10 is carried out from wet cells or dry cells of the microbial cells or disrupted product thereof by using a hydrophilic organic solvent. 13. The process as claimed in any one of Claims 12, wherein the hydrophilic organic solvent is acetone, aeetonitzile, methanol, ethanol, 1- groganol or 2-propanol. 14. The process as claimed in any one of Claims 1 to 11, wherein the extraction of the coenzymes Q10 is carried out from an aqueous suspension of the microbial cells or disrupted product thereof by using a hydrophobic organic solvent. 15. The process as claimed in Claim 14, wherein the hydrophobic organic solvent is a hydrocarbon, a fatty acid ester or an ether. 16. The process as claimed in Claim 14 or 15, wherein the hydrophilic organic solvent is used as an auxiliary solvent in combination with the hydrophobic organic solvent. 17. The process as claimed in Claim 16, wherein the hydrophobic organic solvent is a hydrocarbon, and the hydrophilic organic solvent is an alcohol. 18. The process as claimed in Claim 16, wherein the hydrophobic organic solvent is an aliphatic hydrocarbon, and the hydrophilic organic solvent is a nonohydric alcohol containing 1 to 5 carbon atoms. 19. The process as claimed in Claim 16, wherein the hydrophobic organic solvent is at least one species of hexane and heptane, and the hydrophilic organic solvent is at least one species of methanol, ethanol, 1- propanol and 2-propanol. 20. The process as claimed in any one of Claims 16 to 19, wherein the extraction is carried out under the condition that the hydrophobic organic solvent is contained in 25 to 65% by volume and the hydrophilic organic solvent is contained in 5 to 50% by volume. 21. The process as claimed in any one of Claims 16 to 20, wherein the extraction is carried out by continuous extraction. 22. The process as claimed in any one of Claims 1 to 21, wherein the reduced coenzyme Q10 is contained at a ratio of not less than 70 mole % among the entire coenzymes Q10 wherein in the case that the reduced coenzyme Q10 producing microorganisms are cultured with shaking (amplitude: 2 cm, 310 reciprocation/min) at 25°C for 72 hours in 10 mL of a culture medium [(glucose: 20 g, peptone: 5 g, yeast extract: 3g, malt extract: 3 g)/L, pH: 6.01 using a test tube (inner diameter: 21 rnm, entire 35 length: 200 m), the obtained broth is optionally concentrated, the obtained solution is vigorously shaken for 3 minutes using 10 parts by volume of glass beads (425 to 600 m) to disrupt the microorganisms under a nitrogen atmosphere in the concomitant presence of 3 parts by volume of isopropanol and 18.5 parts by volume of n-hexane relative to 10 parts by volume of the broth, and the prepared hydrophobic organic solvent phase (n-hexane phase) is analyzed by HPLC. 23. The process as claimed in any one of Claim 22, wherein the reduced coenzyme Q10 producing microorganisms have not less than 1 pgimL of a productivity of reduced coenzyme Q10 per unit culture medium when measured by HPLC under the condition as claimed in Claim 22. 24. The process as claimed in any one of Claims 1 to 23, wherein the microorganisms are microorganisms of the genus Agrobacterium, the genus Aspergillus, the genus Acetobacter, the genus Aminobacter, the genus Agromonas, the genus Acidiphilium, the genus Bulleromyces, the genus Bullera, the genus Brevundimonas, the genus Cryptococcus, the genus Chionosphaera, the genus Candida, the genus Cerinosterus, the genus Exisophiala, the genus Exobasidium, the genus Fellomyces, the genus Filobasidiella, the genus Filobasidium, the genus Geotrichum, the genus Graphiola, the genus Gluconobacter, the genus Kockovaella, the genus Kurtzmanomyces, the genus Lalaria, the genus Leucosporidium, the genus Legionella, the genus Methylobacterlurn, the genus Mycoplana, the genus Oosporidium, the genus Pseudomonas, the genus Psedozyma, the genus Paracoccus, the genus Petromyc, the genus Rhodotorula, the genus Rhodos-oridium, the genus Rhizomonas, the genus Rhodobium, the genus Rhodoplanes, the genus Rhodopseudomonas, the genus Rhodobacter, the genus Sporobolomyces, the genus Sporidiobolus, the genus Saitoella, the genus Schizosaccharomyces, the genus Sphingomonas, the genus Sporotrichum, the genus Sympodiomycopsis, the genus Steriamatosporidium, the genus Tapharina, the genus Tremella, the genus Trichosporon, the genus Tilletiaria, the genus Tilletia, the genus Tolyposporium, the genus Tilletiopsis, the genus Ustilago, the genus Udeniomyce, the genus Xanthophllomyces, the genus Xanthobacter, the genus Paecilomyces, the qenus Acremonium, the genus Hyhomonus, or the genus Rhizobium. 25. The process as claimed in any one of Claims 1 to 24, wherein the obtained oxidized coenzyme Q10 is purified optionally and crystallized to obtain an oxidized coenzyme Q10 crystal. The present invention discloses a process for producing the oxidized coenzyme Q10 which comprises culturing reduced coenzyme Q10 producing microorganisms in a culture medium containing a carbon source, a nitrogen source, a phosphorus source and a micronutrient to obtain microbial cells containing reduced coenzyme Q10 at a ratio of not less than 70 mole % among the entire coenzymes Q10, optionally disrupting the microbial cells; and either oxidizing thus-produced reduced coenzyme Q10 to oxidized coenzyme Q10 using an oxidizing agent and then extracting the resultant by an organic solvent, or extracting thus-producing reduced coenzyme Q10 by an organic solvent, purifying optionally and oxidizing the resultant to oxidized coenzyme Q10 using an oxidizing agent.

Full Text

THIS APPLICATION HAS BEEN DIVIDED OUT OF INDIAN
APPLICATION NO. 00918/KOLNP/2004
TECHNICAL FIELD
The present invention relates to
a process for producing the reduced coenzyme Qio
represented by the following formula (I):

More particularly, the present invention relates to
a process for producing reduced coenzyme Q10
which comprises culturing reduced coenzyme Q10-
producing microorganisms to obtain microbial cells
containing reduced coenzyme Q10 at a ratio of not less than
70 mole % among the entire coenzymes Q10,
optionally disrupting the microbial cells and
recovering thus-produced reduced coenzyme Q10.
The present invention also relates to a process for
producing oxidized coenzyme Q10 which comprises either
recovering oxidized coenzyme Q10 after oxidizing the above-

nentioned microbial cells or disrupted product thereof, or
recovering reduced coenzyme Q10 from the above-mentioned
nicrobial cells or disrupted product thereof to oxidize
thus-obtained reduced coenzyme Q10 thereafter.
BACKGROUND ART
The reduced coenzyme Q10 (I) and the oxidized
coenzyme Q10 (II) are mitochondrial electron transport
system-constituting factors in cells of a living body of
luman and deal with ATP production by working as electron
carriers in oxidative phosphorization reactions.
Conventionally, oxidized coenzyme Q10 has been widely
ised for supplementary nutrient foods and cosmetic products
in addition to pharmaceutical products as a
pharmaceutically and physiologically effective substance
for a variety of diseases.
On the other hand, reduced coenzyme Q10 has not so
nuch drawn attention so far; however, in these years, there
las been reported that reduced coenzyme Q10 is more
affective in various applications than oxidized coenzyme
Q10.
For example, japanise Kukai Publication Hei-10-330251
discloses an antihypercholesterolemia agent having
axcellent cholesterol reducing function, an
iritihyperlipeuiia agent, and an agent for curing and
preventing arteriosclerosis which contain reduced coenzyme
Qio as an active ingredient. In addition, Japanese Kokai
Publication Hei-10-109933 discloses a pharmaceutical
composition excellent in oral absorbability comprising
coenzyme Q10 including reduced coenzyme Q10 as an active
ingredient.
Furthermore, reduced coenzyme Q10 is effective as an
antioxidant and a radical scavenger. R. Stocker, et al.
nave reported that reduced coenzyme Qio prevented
peroxidation of human LDL more efficiently than a-

tocopherol, lycopene and (3-carotene (Proceedings of the
NFational Academy of Science of the United States of America,
vol. 88, pp. 1646-1650, 1991) .
It has been known that oxidized coenzyme Q10 and
reduced coenzyme Q10 are in a certain type of equilibrium
in a living body and that oxidized coenzyme Q10/ reduced
coenzyme Qio absorbed in the living body are mutually
reduced/oxidized.
Reduced coenzyme Q10 is supposedly produced by a
chemical synthesis method, similarly to the process for
producing oxidized coenzyme Q10. But the synthesis process
is supposed to be complicated, risky and costly. Moreover,
in the case of chemical synthesis methods, it will be
necessary to minimize the subgeneraLion and contamination
of a (Z)-isomer, which is suspiciously unsafe (Biomedicai
and Clinical Aspects of Coenzyme Q, vol. 3, pp. 19-30,
1981) . Europe Pharmacopoeia regulates that a content of
(Z)-isomer in oxidized coenzyme Q10 must be not more than
0.1%.
As another process for producing reduced coenzyme Q10,
it can be supposed a method of utilizing microbial cells,
that is, a method for separating and recovering reduced
coenzyme Q10 from reduced coenzyme Q10-producing
microorganisms. However, the reduced coenzyme Q10 produced
by the microbial cells of the above mentioned
microorganisms contains a large amount of oxidized coenzyme
Q10, and the separation and recovery of reduced coenzyme Q10
by a conventional method results in high cost.
The following are documents describing the presence
of reduced coenzyme Q10 in microbial cells and there have
been known the following examples of bacteria.
(1) An example describing that at lowest 5 to 10% by weight
and at highest 30 to 60% by weight of reduced coenzyme Q10
are present among the entire coenzymes Q10 in culture cells
of photosynthesis bacteria (Japanese Kokai Publication Sho-

57-70834).
(2) An example describing that the genus Pseudomonas is
subjected to thermal extraction by an organic solvent in
the presence of sodium hydroxide and pyrogallol, and the
resultant is treated with 5% sodium hydrosulfite solution,
and further dehydrated and concentrated to collect an
acetone-soluble portion, and an oil containing reduced
coenzyme Q10 is obtained (Japanese Kokai Publication Sho-
60-75294).
Both of the above (1) and (2) aim to convert a
mixture of the obtained reduced coenzyme Q10 and oxidized
coenzyme Q10 or the obtained reduced coenzyme Q10 into
oxidized coenzyme Q10 by further oxidation. Thus, reduced
coenzyme Q10 is only described as an intermediate substance
in producing oxidized coenzyme Q10.
In the above (1), photosynthesis bacteria are used,
the culture of which is complicated. Furthermore, in the
microbial cells of the above-mentioned microorganisms, when
the production of reduced coenzyme Q10 is aimed at, it
cannot be said that the ratio of reduced coenzyme Q10 among
the entire coenzymes Q10 is sufficient.
The above (2) comprises a process of converting
oxidized coenzyme Q10 contained in a hexane phase into
reduced coenzyme Q10 by sodium hydrosulfite, a reducing
agent (see Example 3 in Japanese Kokai Publication Sho-60-
75294). Thus, the ratio of reduced coenzyme Q10 among the
entire coenzymes Q10 in the microbial cells is not clear.
Furthermore, in both of the above (1) and (2), the
production amount of coenzymes Q in culture are not
described.
As described above, microbial cells containing
reduced coenzyme Q10 at high ratio have not been reported
yet. Still less, it has not been known a fermentation
production of reduced coenzyme Q10 on the industrial scale,
that is, a method comprising culturing microorganisms to

obtain microbial cells containing reduced coenzyme Q10 at
high ratio among the entire coenzymes Q10, and recovering
reduced coenzyme Q10 to obtain high-purity reduced coenzyme
Q10.
Under such circumstances, if a method for obtaining a
large quantity of coenzyme Q10 containing reduced coenzyme
Q10 at high ratio by culturing microorganisms ' is found, it
can be a highly useful method for producing reduced
coenzyme Q10.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a
process for producing reduced coenzyme Q10 safely and
efficiently on the industrial scale by culturing reduced
coenzyme Q10-producing microorganisms for obtaining
microbial cells containing reduced coenzyme Q10 at high
ratio and suitably recovering reduced coenzyme Q10 from the
microbial cells.
It is another object of the present invention to
provide a process for producing oxidized coenzyme Q10 in
simple processes by culturing reduced coenzyme Q10-
producing microorganisms for obtaining microbiai cells
containing reduced coenzyme Q10 at high ratio, and
oxidizing the reduced coenzyme Q10 obtained from the
microbiai cells as an intermediate substance in producing
oxidized coenzyme Q10.
That is, the present invention relates to
a process for producing the reduced coenzyme Q10
represented by the following formula (I):

which comprises culturing reduced coenzyme Q10-
producing microorganisms in a culture medium containing a
carbon source, a nitrogen source, a phosphorus source and a
micronutrient to obtain microbial cells containing reduced
coenzyme Q10 at a ratio of not less than 70 mole % among
the entire coenzymes Q10,
optionally disrupting the microbial cells and
extracting thus-produced reduced coenzyme Q10 by an
organic solvent.
Furthermore, the present invention also relates to
a process for producing the oxidized coenzyme Q10
represented by the following formula (II):

which comprises culturing reduced coenzyme Q10-
producing microorganisms in a culture medium containing a
carbon source, a nitrogen source, a phosphorus source and a
micronutrient to obtain microbial cells containing reduced
coenzyme Q10 at a ratio of not less than 70 mole % among
the entire coenzymes Q10,
optionally disrupting the microbial cells; and
either oxidizing thus-produced reduced coenzyme Q10
to oxidized coenzyme Q10 and then extracting the resultant

y an organic solvent, or extracting thus-produced reduced
coenzyme Q10 by an organic solvent, purifying optionally
md oxidizing the resultant to oxidized coenzyme Q10.
According to the processes of the present invention,
ceduced coenzyme Q10 can be produced cheaply on the
industrial scale by considerably simple steps comprising
culturing microorganisms and recovering reduced coenzyme
Q10 In addition, oxidized coenzyme Q10 can also be
produced by simple processes. Moreover, these coenzymes Q10
produced by microorganisms basically do not contain (Z)-
isomers thereof, and (all-E) isomers thereof can be
obtained, which are same as those contained in meat, fish,
etc.
DETAILED DISCRIPTION OF THE INVENTION
In the present invention, at first, reduced coenzyme
Q10-producing microorganisms are cultured to obtain
microbial cells containing reduced coenzyme Q10 at a ratio
of not less than 70 mole %, preferably not less than 75
mole %, among the entire coenzymes Q10 (fermentation).
The microbial cells containing reduced coenzyme Q10
aL such high ratio among the entire coenzymes Q10 can be
basically obtained by culturing microorganisms capable of
producing reduced coenzyme Q10 at a ratio of not less than
7 0 mole %, preferably not less than 7 5 mole %, among the
entire coenzymes Q10.
How much ratio the microorganisms can produce reduced
coenzyme Q10 among the entire coenzymes Q10 can be evaluated,
for example, by a method comprising culturing the
microorganisms with shaking (amplitude: 2 cm, 310
reciprocation/min) at 25°C for 72 hours in 10 mL of a
culture medium [(glucose: 20 g, peptone: 5 g, yeast
extract: 3g, malt extract: 3 g)/L, pH: 6.0] using a test
tube (inner diameter: 21 mm, entire length: 200 mm).
Although the preferable culture conditions for the

fermentation production on the industrial scale will be
iescribed later, the above-mentioned culture condition is
one method for standardizing the ratio of reduced coenzyme
Q10 produced, which microorganisms have as its ability, so
as to reflect the ratio within the range without having
significant inaccuracies.
Under the above-mentioned culture condition, it is
preferable to use microbial cells wherein a content of
reduced coenzyme Q10 is at a ratio of not less than 70
mole %, preferably not less than 75 mole %, among the
entire coenzymes Q10, for the present invention. It is
still more preferable to use microorganisms having a
productivity of reduced coenzyme Q10 per unit culture
medium of generally not less than 1 g/mL, preferably not
less than 2 (.g/mL under the above-mentioned culture
condition.
The above-mentioned content of reduced coenzyme Q10
and ratio of reduced coenzyme Q10 among the entire
coenzymes Q10 can be confirmed by physically disrupting the
microbial cells, extracting coenzyme Q10 from thus-obtained
cells by an organic solvent and performing HPLC analysis.
Specifically, the measurement can be carried out according
to the following procedures:
(1) The broth of microorganism is optionally concentrated,
10 parts by volume of the broth are displaced to a screw
cap test tube (inner diameter: 16.5 mm, entire length: 130
mm), and 10 parts by volume of glass beads are added (425
to 600 urn, manufactured by SIGM7A Co.);
(2) 3 parts by volume of isopropanol and 18.5 parts by
volume of n-hexane relative to 10 parts by volume of the
broth are added under a nitrogen atmosphere;
(3) microbial cell disruption and extraction are carried
out by vigorously shaking of the mixture for 3 minutes
under a nitrogen atmosphere; and
(4) the obtained hydrophobic organic solvent phase (n-

hexane phase) is evaporated (bath temperature: 40°C) under
reduced pressure to analyze the resultant by HPLC.
Column: YMC-Pack 4.6 x 250 mm (manufactured by YMC.
Co., Ltd.)
Mobile phase: methanol/n-hexane = 85/15
Flow rate: 1 mL/min,
Detection: UV 275 nm
Retention time: reduced coenzyme Q10 13.5 min
oxidized coenzyme Q10 22.0 min
The above-mentioned measurement method is provided
for the obtained result to reflect the reduced coenzyme Q10
content and the ratio of reduced coenzyme Q10 among the
entire coenzymes Q10 as accurate as possible, and to
standardize the content and the ratio of reduced coenzyme
Q10, which can be guaranteed at the minimum. This method
has been demonstrated, by several experimentations
performed by the present inventors, easy and suitable to be
carried out.
As the above-mentioned reduced coenzyme Q10-producing
microorganisms to be used in the present invention,
bacteria, yeast and fungi may be used without any specific
limitation. As specific examples of the above-mentioned
microorganisms, there may be mentioned, for example,
microorganisms of the genus Agrobacterium, the genus
Aspergillus, the genus Acetobacter, the genus Aminobacter,
the genus Agromonas, the genus Acidiphilium, the genus
Bulleromyces, the genus Bullera, the genus Brevundimonas,
the genus Cryptococcus, the genus Chionosphaera, the genus
Candida, the genus Cerinosterus, the genus Exisophiala, the
genus Exobasidium, the genus Fellomyces, the genus
Filobasidiella, the genus Filobasidium, the genus
Geotrichum, the genus Graphiola, the genus Gluconobacter,
the genus Kockovaella, the genus Kurtzmanomyces, the genus
Lalaria, the genus Leucosporidium, the genus Legionella,
the genus Methylobacterium, the genus Mycoplana, the genus

Dosporidium, the genus Pseudomonas, the genus Psedozyma,
the genus Paracoccus, the genus Petromyc, the genus
Rhodotorula, the genus Rhodosporidium, the genus Rhizomonas,
the genus Rhodobium, the genus Rhodoplanes, the genus
Rhodopseudomonas, the genus Rhodobacter, the genus
Sporobolomyces, the genus Sporidiobolus, the genus
Saitoella/ the genus Schizosaccharomyces, the' genus
Sphingomonas, the genus Sporotrichum, the genus
Sympodiomycopsis, the genus Sterigmatosporidium, the genus
Tapharina. the genus Tremella, the genus Trichosporon, the
genus Tilletiaria, the genus Tilletia, the genus
Tolyposporium, the genus Tilletiopsis, the genus Ustilago,
fhp genus Udeniomyce, the genus Xanthophllomyces, the genus
Xanthobacter, the genus Paecilomyces, the genus Acremonium,
the genus Hyhomonus, and the genus Rhizobium.
In terms of the culture easiness and productivity,
bacteria (preferably nonphotosynthetic bacteria) and yeast
are preferred. As the bacteria, there may be mentioned,
for example, the genus Agrobacterium, the genus
Gluconobacter and the like. As the yeast, there may be
mentioned, for example, the genus Schizosaccharomyces, the
genus Saitoeiia and the like.
As preferable species, there may be mentioned, for
example, Agrobacterium tumefacience IFO13263, Agrobacterium
radiobacter ATCC4718, Aspergiiius ciavatus JCM1718,
Acetobacter xylinum IFO15237, Aminobacter aganouensis
JCM7854, Agromonas oligotrophica JCM1494, Acidiphilium
multivorum JCM8 8 67, Bulleromyces albus IFO1192, Bullera
armeniaca IFO10112, Brevundimonas diminuta JCM2788,
Cryptococcus laurentii IFO0609, Chionosphaera apobasidialis
CBS7430, Candida curvata ATCC10567, Cerinosterus luteoalbus
JCM2923, Exisophiala alcalophila JCM12519, Exobasidium
gracile IFO7788, Fellomyces fuzhouensis IFO10374,
Filobasidiella neoformans CBS132, Filobasidium
capsuloigenum CBS1906, Geotrichum capitatum JCM6258,

Graphiola cylindrica IFO6426, Gluconobacter suboxydans
IFO32 57, Kockovaella imperatae JCM7 82 6, Kurtzmanomyces
nectairei IFO10118, Lalaria cerasi CBS275.28,
Leucosporidium scottii IFO1212, Legionella anisa JCM7573,
Methylobacterium extorguens JCM2 8 02, Mycoplana ramosa
JCM7822, Oosporidium margaritiferum CBS2531, Pseudomonas
denitrificans IAM 12023, Psaudomonas shuylkil.liensis IAM
1092, Psedozyma aphidis CBS517.23, Paracoccus denitrificans
JCM6892, Petromyces alliaceus IFO7538, Rhodotorula glutinis
IFO1125. Rhodotorula minuta IFO03 87, Rhodosporidium
diobovatum ATCC1830, Rhizomonas suberifaciens IFO15212,
Rhodobium orients JCM9337, Rhodoplanes elegans JCM922 4,
Rhodopseudomonas palustris JCM2524, Rhodobacter capsulatus
SB1003, Sporobolomyces holsaticus IFO1034, Sporobolomyces
pararoseus IFO0471, Sporidiobolus johnsonii IFO1840,
Saitoella complicata IFO10748, Schizosaccharomyces pombe
IFO0347, Sphingomonas parapaucimobilis IFO15100,
Sporotrichum cellulophilium ATCC2 04 93, Sympodiomycopsis
paphiopedili JCM8318, Sterigmatosporidium polymorphum
IFO10121, Sphingomonas adhesiva JCM7370, Tapharina
caerulescens CBS351.35, Tremella mesenterica ATCC24438,
Trichosporon cutaneum IFO1198, Tilletiaria anomaia
CBS4 3 6.72, Tilletia caries JCM17 61, Tolyposporium buiiatum
JCM2006, Tilletiopsis washintonesis CBS544, Ustilago
esculenta IFO9887, Udeniomyces megalosporus JCM5269,
Xanthophllomyces dendrorhous IFO10129, Xanthobacter flavus
JCM1204, Paecilomyces lilacinus ATCC10114, Acremonium
chrysogenum ATCC11550, Hyphomonas hirschiana ATCC33886,
Rhizobium meliloti ATCC9930, and the like.
As the reduced coenzyme Q10-producing microorganisms,
not only the wild species of the above-mentioned
microorganisms but also microorganisms in which the
transcription and translation activities of the genes
relevant to the biosynthesis of reduced coenzyme Q10 in the
above-mentioned microorganisms, or the enzyme activity of

the expressed protein are modified or improved can be used
preferably, for example.
As the means for modifying or improving the
transcription and translation activities of the genes or
the enzyme activity of the expressed protein, there may be
mentioned gene recombination (including gene improvement,
amplification and destruction by itself, external gene
introduction, and gene improvement and proliferation of
thus-introduced external genes) and mutagenesis by mutagens
In particular, the mutagenesis by mutagens is preferred.
The more preferable microorganisms usable for the
present invention are microorganisms containing reduced
coenzyme Q10 at a ratio of not less than 70 mole %,
preferably not less than 75 mole %, more preferably not
less than 80 mole %, still more preferably not less than 85
mole %, and particularly preferably not less than 90 mole %,
among the entire coenzymes Q10 in the case where the above-
mentioned modified or improved microorganisms, preferably
microorganisms mutated by mutagens, are evaluated by the
above-mentioned proliferation method and the measurement
method. In the fermentation production on the industrial
scale, it is preferable to use microorganisms haviny a
productivity of reduced coenzyme Q10 per unit culture
medium of not less than 1 ug/mL, preferably not less than 2
(.g/mL, more preferably not less than 3 |g/mL, still more
preferably not less than 5 .g/mL, particularly preferably
not less than 10 g/mL, much more preferably not less than
15 g/mL, and most preferably not less than 20 ug/mL.
The mutagenesis may be carried out by a single
mutagenesis; however, mutagenesis is preferably carried out
not less than 2 times. That is because it was found that
the productivity of reduced coenzyme Q10 can be improved in
the respective mutagenesis steps. It is needless to say
that the candidates of the microbial cells to be mutated
are, generally, those having a productivity of reduced

coenzyme Q10 as high as possible in the case where the
evaluation is carried out by the above-mentioned
proliferation method and measurement method.
The mutagenesis can be carried out by using optional
and proper mutagens. The term "mutagen" encompasses, in a
board definition, not only chemical agents having
mutagenesis effects, for example, but also treatments such
as UV radiation having mutagenesis effects. As examples of
proper mutangens, there may be mentioned ethyl
methanesulfonate, UV radiation, N-methyl-N'-nitro-N-
nitrosoguanidine, nucleotide base analogues such as
bromouracil, and acridines; however, they are not limited
to these examples.
According to a conventional mutagenesis technique,
successively to the mutagenesis, a proper selection of
microbial cells having high productivity of reduced
coenzyme Q10 is carried out. For that, the culture obtained
from a single colony should be evaluated, for example, by
the above-mentioned proliferation method and measurement
method. Since a reduced coenzyme Qlo crystal forms a white
solid layer or a colorless liquid phase, a productivity of
reduced coenzyme Q10 can be suitably evaluated by the
above-mentioned measurement method at the time of selection
of the colony.
In the processes of the present invention, high
productivity of reduced coenzyme Qlo in the fermentation
production on the industrial scale can be achieved
partially by using the microbial cells containing reduced
coenzyme Q10 at a ratio of not less than 70 mole % among
the entire coenzymes Q10 and, partially, by using the
suitable conditions of culture (fermentation) for
increasing a productivity of reduced coenzyme Q10 per unit
culture medium as described below. It is particularly
preferable to combinedly use suitable microbial cells
described above and the suitable conditions of culture

(fermentation) as described below.
The culture is carried out, in general, in a culture
medium containing major nutrients and micronutrients suited
for microorganism proliferation. As the above-mentioned
nutrients, there may be mentioned, for example, carbon
sources (e.g. hydrocarbons such as glucose, sucrose,
maltose, starch, corn syrup and molasses; alcohols such as
methanol and ethanol), nitrogen sources (e.g. corn steep
liquor, ammonium sulfate, ammonium phosphate, ammonium
hydroxide, urea and peptone), phosphorus sources (e.g.
ammonium phosphate and phosphoric acid) and micronutrients
(e.g. minerals such as magnesium, potassium, zinc, copper,
iron, manganese, molybdenum, sulfuric acid and hydrochloric
acid; vitamins such as biotin, desthiobiotin and vitamin
Bl; amino acids such as alanine and histidine; and natural
raw materials containing vitamins such as yeast extract and
malt extract); however, these are not limitative ones, and
commonly used ones may be used. Incidentally, in natural
components of a culture medium, such as yeast extract,
phosphorus sources such as phosphates are contained. The
above-mentioned nutrients can be appropriately used in
combination.
The culture is generally carried out at a temperature
range of 15 to 45°C, preferably 20 to 37°C. If it is below
15°C, the proliferation speed of microorganisms tends to be
too slow to allow the industrial production and at high
temperatures exceeding 45°C, the viability of
microorganisms tends to be easily hindered.
In general, the culture is carried out at a pH range
of 4 to 9, preferably 5 to 8 . If the pH is not more than 3
or not less than 10, proliferation of microorganisms tends
to be easily inhibited.
In the fermentation production on the industrial
scale, although it depends on the microorganism species,
the concentration of the carbon sources (including the

produced alcohols) during the culture is preferably
controlled to a concentration that no adverse effects are
substantially caused on the productivity of reduced
coenzyme Q10. Accordingly, it is preferable to control the
culture so as to have the concentration of the carbon
sources that no adverse effects are substantially caused on
the productivity of reduced coenzyme Q10, that is,
generally to not more than 20 g/L, preferably not more than
5 g/L, and more preferably not more than 2 g/L in the broth.
To control the concentration of the carbon sources, a
fed batch culture method is preferably used. The carbon
source concentration in the broth can be controlled by
adjusting the supply of nutrient sources (especially carbon
sources) based on the culture control indexes such as pll,
the dissolved oxygen concentration (DO) or the remaining
saccharide concentration. Although it depends on the
microorganism species, the supply of the nutrient sources
nay be started from the initial stage of the culture or
during the culture. The supply of the nutrient sources may
be continuous or intermittent. Incidentally, in supplying
the nutrient sources, it is preferable to supply the above-
mentioned carbon sources Lo the culture medium separately
from other components.
The culture can be completed at the point when a
desired amount of reduced coenzyme Q10 is produced. The
culture duration is not particularly limited and it is
generally 20 to 200 hours.
The above-mentioned culture is generally carried out
aerobically. The term "aerobically" means a condition that
oxygen is supplied so as not to cause oxygen limitation
(oxygen deficiency) during the culture, and preferably a
condition that oxygen is supplied sufficiently so as not to
cause substantial oxygen limitation during the culture.
The culture is carried out generally under an aeration
condition, preferably under an aeration and stirring

condition.
By using the above-mentioned microorganisms and
culture conditions, it becomes possible to obtain microbial
cells containing reduced coenzyme Q10 at a ratio of not
less than 70 mole %, preferably not less than 75 mole %
among the entire coenzymes Q10. Furthermore, the
productivity of reduced coenzyme Q10 of as high as not less
than 1 g/mL, preferably not less than 2 (g/mL, and still
more preferably not less than 3 |xg/mL can be obtained.
Next, recovery of the reduced coenzyme Q10 produced
by the above-mentioned culture will be described.
In the present invention, an efficient production of
reduced coenzyme Q10 on the industrial scale is made to be
possible partially by the above-mentioned suitable culture
and partially by the suitable recovery process of reduced
coenzyme Q10 as described below.
Recovery of reduced coenzyme Q10 is carried out by
extraction from the microbial cells obtained by the above-
mentioned culture using an organic solvent.
In the extraction, cells can be disrupted optionally.
The cell disruption contributes to the efficient extraction
of the reduced coenzyme Q10 produced and accumulated in
cells. It is needless to say that the cell disruption and
extraction can be carried out at the same time.
Incidentally, "disruption" in the present invention
may be carried out to the extent that the surface structure
such as a cell wall is broken so as to make extraction of
reduced coenzyme Q10 possible; therefore, it is not
necessary that microbial cells are torn or fragmentated.
The above-mentioned cell disruption is not
necessarily required in the case of bacteria. However, in
the case of yeast or fungi, the cell disruption is
generally required and, when cells are not disrupted, it
becomes difficult to efficiently recover the reduced
coenzyme Q10 produced and accumulated in the cells.

The above-mentioned disruption of microbial cells can
De carried out by the following one or several disruption
nethods in optional order. As the disruption method, there
nay be mentioned, for example, a physical treatment, a
chemical treatment., an enzymic treatment as well as a
heating treatment, an autolysis, an osmolysis, a
plasmoptysis and the like.
The above-mentioned physical treatment can be carried
out, for example, by using a high pressure homogenizer, an
ultrasonic homogenizer, a French press, a ball mill and the
like or using them in combination.
The above-mentioned chemical treatment can be carried
out, for example, by using an acid (preferably a strong
acid) such as hydrochloric acid and sulfuric acid, a base
(preferably a strong base) such as sodium hydroxide and
potassium hydroxide and the like or using them in
combination.
The above-mentioned enzymic treatment can be carried
out, for example, by using lysozyme, zymolyase, glucanase,
Novozyme, protease, cellulase and the like or by using them
appropriately in combination.
The dbove-mentioned heating treatment can be carried
out, for example, by heating to the temperature range of 60
to 100°C for about 30 minutes to 3 hours.
The above-mentioned autolysis can be carried out, for
example, by treatment with a solvent such as ethyl acetate.
The osmolysis or the plasmoptysis for disrupting
cells by treating cells with a solution having a different
salt concentration from that in the cells are often
combinedly used with the above-mentioned physical treatment,
chemical treatment, enzymic treatment, heating treatment,
autolysis and/or the like since the above lytic method
alone is insufficient in the disruption effect.
As the cell disruption method as a pretreatment of
extraction and recovery of reduced coenzyme Q10, among the

above-mentioned disruption methods, the physical treatment,
the chemical treatment (particularly, an acid treatment and
preferably the one with a strong acid (e.g. an acid having
a pKa value of not more than 2.5 in the form of an aqueous
solution) under the condition that reduced coenzyme Q10 is
protected from an oxidation reaction as described below)
and the heating treatment are preferred. From the
viewpoint of disruption efficiency, the physical treatment
is more preferred.
A conventional cell disruption method and coenzyme
Q10 extraction method, specifically, a method comprising
extracting coenzyme Q10 by an organic solvent in the
presence of sodium hydroxide and pyrogallol has problems in
terms of cost, waste treatment, safety in effective
utilization of waste microorganisms (waste cells) such as
recovery of protein, and the like. However, the cell
disruption method, particularly the physical treatment
method of the present invention, does not cause
subgeneration of a large quantity of salts by
neutralization, and is a suitable method from a viewpoint
of the waste treatment and the effective utilization of
waste microorganisms (wasle cells).
The form of the microbial cells to be used for the
above-mentioned cell disruption may be a broth, a
concentrated broth, microbial cells collected as wet cells
from the broth, a product obtained by washing them, a
suspension of the wet cells in a solvent (including, for
example, water, physiological saline solution, buffers and
the like), dry cells obtained by drying the above-mentioned
wet cells, a suspension of the dry cells in a solvent
(including, for example, water, physiological saline
solution, buffers and the like), and the like. Preferred
is an aqueous suspension of microbial cells, and in terms
of operability and the like, more preferred are the broth,
the concentrated broth, and the product obtained by washing

them.
The form of the above-mentioned microbial cells or
disrupted product thereof to be used for extraction and
recovery of reduced coenzyme Q10 is, similarly as described
above, not particularly limited and may be wet cells/dry
cells of the microbial cells/disrupted product thereof.
Preferably, it is an aqueous suspension of the microbial
cells or disrupted product thereof, and more preferably the
broth, the concentrated and/or washed broth, or solutions
obtained by disrupting them (each of them is an aqueous
suspension).
The cell concentration in the above-mentioned
suspension of the microbial cells or disrupted product
thereof is not particularly limited and is generally 1 to
25% by weight on the basis of dry weight. Preferably, it
is 10 to 20% by weight in terms of cost.
Reduced coenzyme Q10 can be recovered by extracting
the microbial cells and disrupted product thereof obtained
in such a manner by an organic solvent.
As the organic solvent to be used for the extraction,
there may be mentioned hydrocarbons, fatty acid esters,
ethers, alcohols, fatty acids, ketones, nitrogen compounds
(including nitriles and amides), sulfur compounds and the
like.
Particularly, in extracting reduced coenzyme Q10, in
terms of protection from oxidation by a molecular oxygen,
at least one species of hydrocarbons, fatty acid esters,
ethers, and nitriles is preferably used. Among them,
hydrocarbons and fatty acid esters are particularly
preferable, and hydrocarbons are most preferable.
On the industrial production scale, complete oxygen
elimination is very difficult to be achieved and,
furthermore, fairly long periods of time are required for
individual operations, unlike laboratory scale production,
so that residual oxygen exerts a great adverse effect. The

oxidation in question is directly connected to a
subgeneration of oxidized coenzyme Q10 from reduced
coenzyme Q10. Accordingly, use of the above-mentioned
organic solvent (such as hydrocarbons, fatty acid esters,
ethers, and nitriles) with high oxidation prevention effect
in the extraction of reduced coenzyme Q10 assists an
efficient extraction.
The hydrocarbons are not particularly restricted, but
there may be mentioned, for example, aliphatic hydrocarbons,
aromatic hydrocarbons, halogenated hydrocarbons, and the
like. Preferred are aliphatic hydrocarbons and aromatic
hydrocarbons, and more preferred are aliphatic hydrocarbons.
The aliphatic hydrocarbons are not particularly
restricted, and may be cyclic or acyclic, or saturated or
unsaturated. However, generally, saturated ones are
preferably used. Usually, ones containing 3 to 20 carbon
atoms, preferably 5 to 12 carbon atoms, and more preferably
5 to 8 carbon atoms are used. As specific examples, there
may be mentioned, for example, propane, butane, isobutane,
pentane, 2-methylbutane, hexane, 2-methylpentane, 2,2-
dimethylbutane, 2,3-dimethylbutane, heptane, heptane
isomers (e.g. 2-methylhexane, 3-methylhexane, 2,3-
dimethylpentane, 2,4-dimethylpentane), octane, 2,2,3-
trimethylpentane, isooctane, nonane, 2,2,5-trimethylhexane,
decane, dodecane, 2-pentene, 1-hexene, 1-heptene, 1-octene,
1-nonene, 1-decene, cyclopentane, methylcyclopentane,
cyclohexane, methylcyclohexane, ethylcyclohexane, p-
menthane, cyclohexene, and the like. Preferred are pentane,
2-methylbutane, hexane, 2-methylpentane, 2,2-dimethylbutane,
2,3-dimethylbutane, heptane, heptane isomers (e.g. 2-
methylhexane, 3-methylhexane, 2,3-dimethylpentane, 2,4-
dimethylpentane), octane, 2, 2, 3-trimethylpentane, isooctane,
nonane, 2,2,5-trimethylhexane, decane, dodecane,
cyclopentane, methylcyclopentane, cyclohexane,
methylcyclohexane, ethylcyclohexane, p-menthane, and the

like. More preferred are pentane, 2-methylbutane, hexane,
2-methylpentane, 2,2-dimethylbutane, 2, 3-dimethylbutane,
heptane, heptane isomers (e.g. 2-methylhexane, 3-
methylhexane, 2,3-dimethylpentane, 2,4-dimethylpentane),
octane, 2,2,3-trimethylpentane, isooctane, cyclopentane,
methylcyclopentane, cyclohexane, methylcyclohexane,
ethylcyclohexane, and the like.
Generally, heptanes, not only heptane but also
heptane isomers such as methylcyclohexane having 7 carbon
atoms and a mixture thereof are preferably used. More
preferred are pentanes (e.g. pentane and the like) having 5
carbon atoms, hexanes (e.g. hexane, cyclohexane and the
like) having 6 carbon atoms, and heptanes (e.g. heptane,
mRthylcyclohexane and the like) having 7 carbon atoms.
Particularly preferred are heptanes (e.g. heptane,
methylcyclohexane and the like) in terms of especially high
protection effect from oxidation, and the most preferred is
heptane.
The aromatic hydrocarbons are not particularly
restricted, but generally ones containing 6 to 20 carbon
atoms, preferably 6 to 12 carbon atoms, and more preferably
7 to 10 carbon atoms are used. As specific examples, there
may be mentioned, for example, benzene, toluene, xylene, o-
xylene, m-xylene, p-xylene, ethylbenzene, cumene,
mesitylene, tetralin, butylbenzene, p-cymene,
cyclohexylbenzene, diethylbenzene, pentylbenzene,
dipentylbenzene, dodecylbenzene, styrene, and the like.
Preferred are toluene, xylene, o-xyiene, m-xylene, p-xylene,
ethylbenzene, cumene, mesitylene, tetralin, butylbenzene,
p-cymene, cyclohexylbenzene, diethylbenzene, pentylbenzene
and the like. More preferred are toluene, xylene, o-xylene,
m-xylene, p-xylene, cumene, tetralin and the like, and most
preferred is cumene.
The halogenated hydrocarbons are not particularly
restricted, and may be cyclic or acyclic, or saturated or

unsaturated. However, acyclic ones are preferably used in
general. Usually, more preferred are chlorinated
hydrocarbons and fluorinated hydrocarbons, and chlorinated
hydrocarbons are still more preferred. Additionally, ones
containing 1 to 6 carbon atoms, preferably 1 to 4 carbon
atoms, and more preferably 1 to 2 carbon atoms are suitably
used. As specific examples, for example, there may be
mentioned dichloromethane, chloroform, carbon tetrachloride,
1, 1-dichloroethane, 1,2-dichloroethane, 1,1,1-
trichloroethane, 1,1.2-trichloroethane, 1,1,1,2-
tetrachloroethane,- 1, 1, 2, 2-tetrachloroethane,
pentachloroethane, hexachloroethane, 1,1-dichloroethylene,
1, 2-dichloroethylene, trichloroethylene,
tetrachloroethylene. 1.2-dichloropropane, 1,2,3-
trichloropropane, chlorobenzene, 1,1,1,2-tetrafluoroethane,
and the like. Preferred are dichloromethane, chloroform,
carbon tetrachloride, 1,1-dichloroethane, 1,2-
dichloroethane, 1,1,1-trichloroethane, 1,1,2-
trichloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene,
trichloroethylene, chlorobenzene, 1,1,1,2-tetrafluoroethane,
and the like. More preferred are dichloromethane,
chloroform, 1,2-dichloroethylene, trichloroethylene,
chlorobenzene, 1,1,1,2-tetrafluoroethane and the like.
The fatty acid esters are not particularly restricted,
but there may be mentioned, for example, propionates,
acetates, formates, and the like. Preferred are acetates
and formates, and more preferred are acetates. Ester
functional groups thereof are not particularly restricted,
but, in general, preferred are alkyl esters having 1 to 8
carbon atoms and aralkyl esters having 7 to 12 carbon atoms,
more preferred are alkyl esters having 1 to 6 carbon atoms,
and still more preferred are alkyl esters having 1 to 4
carbon atoms.
As specific examples of the propionates, there may be
mentioned, for example, methyl propionate, ethyl propionate,

butyl propionate, isopentyl propionate, and the like.
Preferred are ethyl propionate and the like.
As specific examples of the acetates, there may be
mentioned, for example, methyl acetate, ethyl acetate,
propyl acetate, isopropyl acetate, butyl acetate, isobutyl
acetate, sec-butyl acetate, pentyl acetate, isopentyl
acetate, sec-hexyl acetate, cyclohexyl acetate, benzyl
acetate, and the like. Preferred are methyl acetate, ethyl
acetate, propyl acetate, isopropyl acetate, butyl acetate,
isobutyl acetate, sec-butyl acetate,- pentyl acetate,
isopentyl acetate, sec-hexyl acetate, cyclohexyl acetate,
and the like. More preferred are methyl acetate, ethyl
acetate, prcpyl acetate, isopropyl acetate, butyl acetate,
acetate.
As specific examples of the formates, there may be
mentioned, for example, methyl formate, ethyl formate,
propyl formate, isopropyl formate, butyl formate, isobutyl
formate, sec-butyl formate, pentyl formate, and the like.
Preferred are methyl formate, ethyl formate, propyl formate,
butyl formate, isobutyl formate, pentyl formate, and the
like. Most preferred is ethyl formate.
The ethers are not particularly restricted, and may
be cyclic or acyclic, or saturated or unsaturated. But
saturated ones are preferably used in general. Generally,
ones containing 3 to 20 carbon atoms, preferably 4 to 12
carbon atoms and more preferably 4 to 8 carbon atoms are
used. As specific examples, there may be mentioned, for
example, diethyl ether, methyl tert-butyl ether, dipropyl
ether, diisopropyl ether, dibutyl ether, dihexyl ether,
ethyl vinyl ether, butyl vinyl ether, anisol, phenetole,
butyl phenyl ether, methoxytoluene, dioxane, furan, 2-
methylfuran, tetrahydrofuran, tetrahydropyran, ethylene
glycol dimethyl ether, ethylene glycol diethyl ether,
ethylene glycol dibutyl ether, ethylene glycol monomethyl

ether, ethylene glycol monoethyl ether, ethylene glycol
monobutyl ether, and the like. Preferred are diethyl ether,
methyl tert-butyl ether, dipropyl ether, diisopropyl ether,
dibutyl ether, dihexyl ether, anisol, phenetole, butyl
phenyl ether, methoxytoluene, dioxane, 2-methylfuran,
tetrahydrofuran, tetrahydropyran, ethylene glycol dimethyl
ether, ethylene glycol diethyl ether, ethylene glycol
dibutyl ether, ethylene glycol monomethyl ether, ethylene
glycol monoethyl ether, and the like. More preferred are
diethyl ether, methyl tert-butyl ether, anisol,- dioxane,
tetrahydrofuran, ethylene glycol. monomethyl ether, ethylene
glycol monoethyl ether, and the like. Still more preferred
are dietbvl ether- inethvl tert-butv] ether- anisol - and the
like, and most preferred is methyl tcrt-butyl ether.
The alcohols are not particularly restricted but may
be cyclic or acyclic, or saturated or unsaturated.
Saturated ones are generally preferred, however. Generally,
ones containing 1 to 20 carbon atoms, more preferably 1 to
12 carbon atoms, and still more preferably 1 to 6 carbon
atoms are used. Among them, monohydric alcohols containing
1 to 5 carbon atoms, dihydric alcohols containing 2 to 5
carbon atoms, and trihydric alcohols containing 3 carbon
atoms are preferred.
As specific examples of these alcohols, there may be
mentioned, for example, monohydric alcohols such as
methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-
butanol, isobutyl alcohol, tert-butyl alcohol, 1-pentanol,
2-pentanol, 3-pentanol, 2-methyl-l-butanol.. isopentyl
alcohol, tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl
alcohol, 1-hexanol, 2-methyl-l-pentanol, 4-methyl-2-
pentanol, 2-ethyl-l-butanol, 1-heptanol, 2-heptanol, 3-
heptanol, 1-octanol, 2-octanol, 2-ethyl-l-hexanol, 1-
nonanol, 1-decanol, 1-undecanol, 1-dodecanol, allyl alcohol,
propargyl alcohol, benzyl alcohol, cyclohexanol, 1-
methylcyclohexanol, 2-methylcyclohexanol, 3-

methylcyclohexanol, 4-methylcyclohexanol, and the like;
dihydric alcohols such as 1,2-ethanediol, 1,2-propandiol,
1,3-propandiol, 1,2-butanediol, 1,3-butanediol, 1,4-
butanediol, 2,3-butanediol, 1,5-pentanediol, and the like;
and trihydric alcohols such as glycerol, and the like.
As the monohydric alcohols, preferred are methanol,
ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol,
isobutyl alcohol, tert-butyl alcohol, 1-pentanol, 2-
pentanol, 3-pentanol, 2-methyl-l-butanol, isopentyl alcohol,
tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol,
1-hexanol, 2-methyl-l-pentanol, 4-methyl-2-pentanol, 2-
ethyl-1-butanol, 1-heptanol, 2-heptanol, 3-heptanol, 1-
octanol, 2-octanol, 2-ethyl-l-hexanol, 1-nonanol, 1-decanol,
1-undecanol, 1-dodecanol, benzyl alcohol, cyclohexanol, 1-
methylcyclohexanol, 2-methylcyclohexanol, 3-
methylcyclohexanol, 4-methylcyclohexanol, and the like.
More preferred are methanol, ethanol, 1-propanol, 2-
propanol, 1-butanol, 2-butanol, isobutyl alcohol, tert-
butyl alcohol, 1-pentanol, 2-pentanol, 3-pentanol, 2-
methyl-1-butanol, isopentyl alcohol, tert-pentyl alcohol,
3-methyl-2-butanol, neopentyl alcohol, 1-hexanol, 2-methyl-
i-pentanol, 4-methyi-2-pentanoi, 2-ethyi-l-butanoi,
cyclohexanoi, and the like. Still more preferred are
methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-
butanoi, isobutyi alcohol, tert-butyl alcohol, 1-pentanol,
2-pentanol, 3-pentanol, 2-methyl-l-butanol, isopentyl
alcohol, tert-pentyl alcohol, 3-methyl-2-butanol, neopentyl
alcohol, and the like. Particularly preferred are methanol,
ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol,
isobutyl alcohol, 2-methyl-l-butanol, isopentyl alcohol,
and the like. Most preferred is 2-propanol.
As the dihydric alcohols, preferred are 1,2-
ethanediol, 1,2-propandiol, 1, 3-propandiol, and the like.
Most preferred is 1,2-ethanediol. As the trihydric
alcohols, glycerol is preferred.

As fatty acids, there may be mentioned, for example,
formic acid, acetic acid, propionic acid, and the like.
Preferred are formic acid and acetic acid, and most
preferred is acetic acid.
The ketones are not particularly restricted, and ones
having 3 to 6 carbon atoms are preferably used. As
specific examples, there may be mentioned, fo'r example,
acetone, methyl ethyl ketone, methyl butyl ketone, methyl
isobutyl ketone, and the like. Preferred are acetone and
methyl ethyl ketone, and most preferred is acetone.
The nitriles are not particularly restricted, and may
be cyclic or acyclic, or saturated or unsaturated. However,
saturated ones are preferably used in general. Generally,
ones containing 2 to 20 carbon atoms, preferably 2 to 12
carbon atoms, and more preferably 2 to 8 carbon atoms are
used.
As specific examples, there may be mentioned, for
example, acetonitrile, propiononitrile, malononitrile,
butyronitrile, isobutyronitrile, succinonitrile,
valeronitrile, glutaronitrile, hexanenitrile, heptylcyanide,
octylcyanide, undecanenitrile, dodecanenitrile,
tridecanemtrile, pentadecanenitriie, stearonitriie,
chloroacetonitriie, bromoacetonitrile,
chloropropiononitrile, bromopropiononitrile,
methoxyacetonitriie, methyl cyanoacetate, ethyl
cyanoacetate, tolunitrile, benzonitrile, chlorobenzonitrile,
bromobenzonitrile, cyanobenzoic acid, nitrobenzonitrile,
anisonitrile, phthalonitrile, bromotolunitrile, methyl
cyanobenzoate, methoxybenzonitrile, acetylbenzonitrile,
naphthonitrile, biphenylcarbonitrile, phenylpropiononitrile,
phenylbutyronitrile, methylphenylacetonitrile,
diphenylacetonitrile, naphthylacetonitrile,
nitrophenylacetonitrile, chlorobenzylcyanide,
cyclopropanecarbonitrile, cyclohexanecarbonitrile,
cycloheptanecarbonitrile, phenylcyclohexanecarbonitrile,

tolylcyclohexanecarbonitrile, and the like.
Preferred are acetonitrile, propiononitrile,
succinonitrile, butyronitrile, isobutyronitrile,
valeronitrile, methyl cyanoacetatev ethyl cyanoacetate,
benzonitrile, tolunitrile and chloropropiononitrile. More
preferred are acetonitrile, propiononitrile, butyronitrile
and isobutyronitrile, and most preferred is acetonitrile.
As the nitrogen compounds other than nitriles, there
may be mentioned, for example, amides such as formamide, N-
methylformamide, N, N-dimethylformamide, N,N-
dimethylacetoamide, N-methylpyrrolidone, and nitromethane,
triethylamine, pyridine, and the like.
As the sulfur compounds, there may be mentioned, for
example, dimethyl sulfoxide, sulfolane, and the like.
In selecting the organic solvent to be used from
among the organic solvents mentioned above, such properties
as boiling point and viscosity (e.g. the solvent should
have a boiling point which allows appropriate warming for
increasing solubility and facilitates a solvent removal
from wet masses by drying and solvent recovery from
crystallization filtrates and the like (about 30 to 150°C
at 1 atm), a melting point such that solidification hardly
occurs in handling at room temperature as well as upon
cooling to room temperature or below (not lower than about
0"C, preferably not lower than about 1U°C, more preferably
not lower than about 20°C), and a low viscosity (not higher
than about 10 cp at 20°C and the like)) are preferably
taken into consideration.
The oxidation prevention effect on reduced coenzyme
Q10 in a solvent tends to increase in a highly-concentrated
solution of reduced coenzyme Gh0. Reduced coenzyme Q10
shows high solubility in the above-mentioned organic
solvents with high oxidation prevention effect (e.g.
hydrocarbons, fatty acid esters and the like). The high
solubility makes it possible to handle the highly-

concentrated solution and to promote the oxidation
prevention. A preferable concentration of reduced coenzyme
Q10.for oxidation prevention at the time of extraction is
not particularly limited, but is generally not less than
0.001% by weight, preferably not less than 0.01% by weight,
and more preferably not less than 0.1% by weight as the
concentration of reduced coenzyme Q10 in the above-
mentioned organic solvent. The upper limit is not
particularly limited, however, in general, it is not more
than 10% by weight.
Among the above-mentioned organic solvents, to
extract and recover reduced coenzyme Q10 from wet cells and
dry cells of the microbial cells or disrupted product
thereof, hydrophilic organic solvents are preferably used.
Specifically, there may be mentioned acetone, acetonitrile,
methanol, ethanol, 1-propanol, 2-propanol and the like.
Furthermore, among the above-mentioned organic
solvents, to extract and recover reduced coenzyme Q10 from
the aqueous suspension of the microbial cells or disrupted
product thereof, hydrophobic organic solvents are
preferably used. Use of such solvents assists the removal
ot water-soluble substances derived from microorganisms.
Many of hydrophobic organic solvents have high oxidation
prevention effect as described above, thus are very
advantageous.
As the hydrophobic organic solvents, hydrocarbons,
fatty acid esters and ethers are preferred.
In the case of the above-mentioned extraction
operation, when reduced coenzyme Q10 is extracted from the
aqueous suspension of the microbial cells or disrupted
product thereof, particularly from the aqueous suspension
of the disrupted product, further particularly the case in
which the disrupted product is physically treated, by an
organic solvent, emulsions tend to be partly formed because
of the presence of cell components such as proteins and

phase separation tends to be difficult. Therefore, it
becomes important to suppress the formation of emulsions
mentioned above and to efficiently carry out extraction.
For that, as an extraction solvent, in addition to
the above-mentioned hydrophobic organic solvent, it is
preferable to use a hydrophilic organic solvent as an
auxiliary solvent in combination.
In this case, the hydrophobic organic solvent is not
particularly limited and those mentioned above may be used.
Preferred are hydrocarbons, and more preferred are
aliphatic hydrocarbons. Among the aliphatic hydrocarbons,
those having 5 to 8 carbon atoms are preferably used.
As specific examples of the aliphatic hydrocarbons
containing 5 to 8 carbon atoms, there may be mentioned, for
example, pentane, 2-methylbutane, hexane, 2-methylpentane,
2, 2-dimethylbutane, 2,3-dimethylbutane, heptane, heptane
isomers (e.g. 2-methylhexane, 3-methylhexane, 2,3-
dimethylpentane, 2,4-dimethylpentane), octane, 2,2,3-
trimethylpentane, isooctane, cyclopentane,
methylcyclopentane, cyclohexane, methylcyclohexane,
ethylcyclohexane, and the like. Particularly preferred are
hexane, heptane and methylcyclohexane, and most preferred
are hexane and heptane.
The hydrophilic organic solvent to be used in
combination with the above-mentioned hydrophobic organic
solvent is not particularly limited and those mentioned
above may be used. Preferred are alcohols. Among the
alcohols, monohydric alcohols having 1 to 5 carbon atoms
are preferably used. As specific examples thereof, there
may be mentioned, for example, methanol, ethanol, 1-
propanol, 2-propanol, 1-butanol, 2-butanol, isobutyl
alcohol, tert-butyl alcohol, 1-pentanol, 2-pentanol, 3-
pentanol, 2-methyl-l-butanol, isopentyl alcohol, tert-
pentyl alcohol, 3-methyl-2-butanol, neopentyl alcohol, and
the like. Particularly preferred are methanol, ethanol, 1-

propanol and 2-propanol, and most preferred is 2-propanol.
The amounts of the above-mentioned hydrophilic
organic solvent and hydrophobic organic solvent to be used
are not particularly limited. But preferably, as the
concentration at the time of extraction, the hydrophilic
organic solvent is used in a range of 5 to 50% by volume
and the hydrophobic organic solvent is used in a range of
25 to 65% by volume relative to the total volume of the
entire solution.
In recovering reduced coenzyme Q10, the temperature
at the time of extraction is not particularly limited and
is generally in a range of 0 to 60°C and preferably 20 to
50°C.
As the extraction method, both batch extraction and
continuous extraction (preferably countercurrent multistage
extraction) may be used. However, the continuous
extraction (preferably countercurrent multistage
extraction) is preferable in terms of productivity. The
stirring duration in the batch extraction is not
particularly limited but is generally not less than 5
minutes. The average retention time in the continuous
extraction is not particularly limited but is generally nut
less than 10 minutes.
In recovering reduced coenzyme Q10, it is preferable
to be careful so that reduced coenzyme Q10 is not
decomposed (e.g. so that reduced coenzyme Q10 is not
oxidized to oxidized coenzyme Q10) For that, the above-
mentioned extraction (including cell disruption) is
preferably carried out under an acidic to a weakly basic
condition, and more preferably under an acidic to a neutral
condition. In the case where a pH is used as an index,
although it depends on the contact time, the pH is
generally not more than 10, preferably not more than 9,
more preferably not more than 8, and still more preferably
not more than 7.

By the above-mentioned conditions, an oxidation
reaction can be substantially prevented and, optionally,
more strictly, the above-mentioned cell disruption and/or
extraction are preferably carried out under the condition
that reduced coenzyme Q10 is protected from an oxidation
reaction. It is preferable to carry out at least the
extraction under this condition, and it is more preferable
to carry out the disruption and the extraction under this
condition.
As "the condition that reduced coenzyme Q10 is
protected from an oxidation reaction" means, for example, a
deoxygenized atmosphere (an atmosphere of an inert gas such
as nitrogen gas, carbon dioxide gas, helium gas, argon gas
or hydrogen gas, reduced pressure, a boiling condition); a
high salt concentration condition, for example, preferably
a condition where salts (e.g. inorganic salts such as
sodium chloride and sodium sulfate) are contained in not
less than about 5% in an aqueous phase; the condition in
the presence of a strong acid (e.g. an acid with a pKa
value of not more than 2.5 in an aqueous solution), for
example, in the presence of not less than 0.1 mole % of the
strong acid relative to i mole of reduced coenzyme Q10; and
the condition in the presence of an antioxidant, for
example, in the concomitant presence of ascorbic acid,
citric acid, salts and esters thereof (e.g. not less than
0.1% by weight of them relative to reduced coenzyme Q10) .
There may also be mentioned a reduction condition (a
condition in which oxidized coenzyme Q10 can be converted
into reduced coenzyme Q10) , for example, a condition
involving a contact with a reducing agent such as
dithionous acid.
By the above-mentioned culture (fermentation) and
extraction, reduced coenzyme Q10 can be suitably produced
and recovered. Preferably, an extract containing not less
than 70 mole %, preferably not less than 75 mole % of

reduced coenzyme Q10 among the entire coenzymes Q10 is
obtained.
Thus-obtained extract containing reduced coenzyme Q10
is optionally purified by column chromatography, reduction
treatment, or the like and then subjected to
crystallization to obtain high-purity reduced coenzyme Q10
crystals. Incidentally,- also in this case, a' series of
treatment steps are preferably carried out under "the
condition that reduced coenzyme Q10 is protected from an
oxidation reaction" mentioned above.
In the present invention, oxidized coenzyme Q10 can
be produced by oxidizing the above-mentioned microbial
cells or disrupted product thereof and then extracting
oxidized coenzyme Q10 by an organic solvent, or extracting
reduced coenzyme Q10 from the microbial cells or disrupted
product thereof by an organic solvent, purifying optionally
and oxidizing the resultant to oxidized coenzyme Q10.
The above-mentioned oxidation may be carried out by,
for example, mixing reduced coenzyme Q10 (preferably an
aqueous suspension of the microbial cells or disrupted
product thereof containing reduced coenzyme Q10, an extract
containing reduced coenzyme Q10 or the like) with an
oxidizing agent (e.g. manganese dioxide or the like) and
then, for example, oxidizing the mixture at room
temperature (e.g. 30°C) for not less than 30 minutes.. In
the case where the microbial cells or disrupted product
thereof are oxidized, the extraction operation of oxidized
coenzyme Q10 can be carried out in the same manner as the
above-mentioned extraction operation of reduced coenzyme
Q10. Thereby, oxidized coenzyme Q10 can be efficiently
recovered. Incidentally, it is not necessary to carry out
the recovery of oxidized coenzyme Q10 under "the condition
that reduced coenzyme Q10 is protected from an oxidation
reaction", which is recommended for the recovery of reduced
coenzyme Q10 and the recovery may be carried out in

consideration of general safe operation and the like. The
thus-obtained oxidized coenzyme Q10 may be optionally
purified by column chromatography or the like, and, finally
by conducting crystallization operation, high-purity
oxidized coenzyme Q10 crystals may be obtained.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a schematic diagram of a countercurrent
3-step continuous extraction apparatus used in Example 8.
BEST MODE FOR CARRYING OUT THE INVENTION
The following examples illustrate the present
invention in further detail. These examples are, however,
by no means limitative of the scope of the present
invention.
(Example 1)
Various coenzyme Q10-producing microorganisms shown
in the following Tables 1 to 3 were cultured with shaking
(amplitude: 2 cm, 310 reciprocation/min) at 25°C for 72
hours in 10 mL of culture media [(glucose: 20 g, peptone: 5
g, yeast extract: 3 g, malt extract: 3 g)/L, pH: 6.0] using
test tubes (inner diameter: 21 mm, entire length: 200 mm),
and the obtained broth were optionally concentrated. Under
a nitrogen atmosphere, in the concomitant presence of 3
parts by volume of isopropanol and 18.5 parts by volume of
n-hexane relative to 10 parts by volume of the broth, the
obtained solutions were vigorously shaken for 3 minutes
using 10 parts by volume of glass beads (425 to 600 |im) to
carry out cell disruption and extraction. The obtained
hexane phases were evaporated (at 40°C) under reduced
pressure and analyzed by high performance liquid
chromatography (HPLC) to determine the ratio and the
production amount of reduced coenzyme Q10.

HPLC conditions
Column: YMC-Pack 4.6 x 250 mm (manufactured by YMC.
Co., Ltd.)
Mobile phase: methanol/n-hexane = 85/15
Flow rate: 1 mL/min
Detection: UV 275 ran
The results are shown in Tables 1 to 3. The ratio of
reduced coenzyme Q10 means a mole percentage value of the
ratio of reduced coenzyme Q10 relative to the total of
oxidized coenzyme Q10 and reduced coenzyme Q10 on the basis
of the areas of the peaks of reduced coenzyme Cho and
oxidized coenzyme Cho and the ratio of the mole absorption
coefficients thereof (1 : 7.5).

38
(Example 2)
Rhodotorula glutinis IFO1125 was aerobically cultured
at 25°C for 48 hours in a culture medium (peptone: 5 g,
yeast extract: 3 g, malt extract: 3 g, glucose: 20 g/L, pH:
6.0) . The cells after the culture were collected by
centrifugation and suspended in a phosphoric acid buffer
solution at pH 7 to which N-methyl-N'-nitro-N-
nitrosoguanidine have been added so as to have its
concentration of 200 g/mL. After maintaining the solution
at 2 5°C for 1 hour, the cells were washed for 5 times with
a 0.9% NaCl solution and further suspended in a 0.9% NaCl
solution. The obtained cell suspension was properly
diluted and a colony was to be formed on an agar plate of
the above-mentioned culture medium. The production amount
and the ratio of reduced coenzyme Q10 in the isolated
mutant strain were determined in the same manner as Example
1. The strains having higher production amount and the
ratio of reduced coenzyme Q10 as compared with those of
wild strains was further mutated repeatedly. As the result,
by repeating the mutagenesis for 10 times, mutant strains
with productivity of not less than 15 g/mL were obtained.
In this case, the ratio of reduced coenzyme Qlo was not
less than 80 mole %.
(Example 3)
Saitoella complicata IFO 10748 was aerobically
cultured at 25°C for 72 hours in 10 L of a culture medium
(peptone: 5 g, yeast extract: 3 g, malt extract: 3 g,
glucose: 20 g/L, pH: 6.0). The obtained cells were
disrupted for 2 times at 80 MPa of disruption pressure by a
pressure homogenizer (manufactured by Lanni Co.) sealed
with nitrogen gas to obtain a cell-disrupted solution. The
cell-disrupted solution was subjected to extraction with 30
parts by volume of isopropanol and 40 parts by volume of
hexane for 3 times to obtain an extract. The extraction

ratio was 99%. The ratio of reduced coenzyme Q10 was 97
mole %.
(Example 4)
When mutant strains of Rhodotorula glutinis IFO1125
were aerobically cultured at 25°C in 10 L of a culture
medium (peptone: 10 g, yeast extract: 5 g, malt extract: 3
g, glucose: 20 g/L, pH: 6.0), glucose was fed at the rate
of 4 g/h after the lapse of 48 hours to 96 hours (fed
glucose amount: 190 g). The production amount of reduced
coenzyme Q10 per culture medium was not less than 2 0 ng/mL
and the ratio of reduced coenzyme Q10 was not less than 80
mole %.
(Example 5)
The extract obtained in Example 3 was subjected to
solvent substitution with a hexane solution, the resultant
was adsorbed in a column filled with silica gel and
subjected to development and elution by a solution of n-
hexane/diethyl ether (9/1) to obtain a fraction containing
reduced coenzyme Q10. Furthermore, the fraction was cooled
to 2°C with stirring to obtain a white slurry. All the
above-mentioned operations were carried out in a nitrogen
atmosphere. The obtained slurry was filtered under reduced
pressure, the resulting wet crystals were washed with the
development solution same as used above (the temperature of
the solvent used for washing was 2°C) , and the wet crystals
were dried under reduced pressure (20 to 40°C, 1 to 30
mmHg) to obtain 81 mg of white dry crystals. The purity of
the obtained crystals was 99.9% and the ratio of reduced
coenzyme Q10 was 90 mole %.
(Example 6)
The extract obtained in Example 3 was subjected to
solvent substitution with n-hexane, the resultant was added

with 50 mg of manganese dioxide, and the mixture was
stirred at 30°C for 30 minutes. Thus-obtained reaction
solution was fractionated and purified in the same manner
as Example 5 to obtain 74 mg of high-purity oxidized
coenzyme Q10.
(Example 7)
Saitoella complicata IFO 10748 was aerobically
cultured at 25°C for 72 hours in 500 mL of a culture medium
(peptone: 5 g, yeast extract: 3 g, malt extract: 3 g,
glucose: 20 g/L, pH: 6.0) . The obtained cells were
disrupted for 2 times at 80 MPa of disruption pressure by a
pressure homogenizer (manufactured by Lanni Co.) sealed
with nitrogen gas to obtain a cell-disrupted solution. The
ratio of reduced coenzyme Q10 in the cell-disrupted
solution was 97% relative to the entire coenzymes Q10
including oxidized coenzyme Q10. 200 mL of the cell-
disrupted solution was mixed with isopropanol and n-hexane
at the ratios shown in the first extraction section in the
following Table 4 so as to adjust the total solvent amount
to be 500 mL and the mixtures were stirred at 40°C for 30
minutes to carry out the tirst extraction. After
completion of the extraction, the resultants were kept
standing for 10 minutes and the separated upper layers were
collected. The volume ratios of the lower layers
(residues) relative to the total solution amounts were
defined as indexes of separability and shown as the
interface positions in Table 4.
Furthermore, in order to carry out the second
extraction, the solvent concentrations of the residual
layers were measured and isopropanol and hexane were
further added so as to keep the solvent ratios in the
entire solutions be the ratios shown in the second
extraction section in Table 4. The resulting solutions
were stirred at 40°C for 30 minutes. Then, the solutions

were kept standing for 10 minutes and the upper layers were
collected in the same manner as described above to
determine the solvent concentrations of the residual layers
Isopropanol and hexane were added thereto so as to keep the
solvent ratios in the entire solutions be the ratios shown
in the third extraction section in Table 4, and the
solutions were stirred at 25°C for 30 minutes to carry out
the third extraction.
The ratios of the amounts of reduced coenzyme Q10
contained in the collected upper layers of each of the
first, second and third steps relative to the amount of
reduced coenzyme Q10 contained in the cell-disrupted
solution or the extraction residue before the extraction
were defined as the extraction ratios of reduced coenzyme
Q10 in the respective steps. The calculation results are
shown in Table 4. The integrated extraction ratios of
reduced coenzyme Q10 in the second and third extraction
steps are also shown. In any steps, the static
separability was excellent and the integrated extraction
ratio in the case where extraction was repeated for 3 times
was as high as not less than 90% to show high recovery
ratio. Particularly, in the case where the isopropanol
concentration was adjusted to be not less than 30%, the
recovery ratio was as high as not less than 99%.

(Example 8)
Saitoella complicata IFO 10748 was aerobically
cultured at 25UC for 72 hours in 7bO L of a culture medium
(peptone: 5 g, yeast extract: 3 g, malt extract: 3 g,
glucose: 20 g/L, pH: 6.0). The obtained cells were
disrupted for 2 times at 140 MPa of disruption pressure by
a pressure homogenizer (manufactured by Lanni Co.) sealed
with nitrogen gas to obtain a cell-disrupted solution. The
cell-disrupted solution was subjected to continuous
extraction by a countercurrent 3-step continuous extractioi
apparatus shown in Fig. 1. The capacity of the stirring
tank was 630 L and the capacity of the static separation
tank was 200 L. The cell-disrupted solution was supplied

to the first stirring tank and isopropanol and n-hexane
were supplied to respective steps. The supply amount of
the cell-disrupted solution was 2 L/min and the supply
amounts of isopropanol and n-hexane were adjusted to be 1.3
L/min for isopropanol and 3.7 L/min for n-hexane as the
total of the supply amounts in respective steps. In this
case, the solvent concentration in respective steps was
properly adjusted so that the isopropanol concentration of
5 to 50 v/v % and the n-hexane concentration of 25 to 65
v/v % were kept. The extraction temperature was 40°C and
the treatment duration was 6 hours. At the point after the
lapse of 6 hours, the recovery ratio of reduced coenzyme
Q10 extracted from the cell-disrupted solution was
calculated on the basis of reduced coenzyme Q10 remaining
in the extraction residue in the static separation tank in
the third step to find the recovery ratio of 98.9%. The
static separation was well carried out during the entire
operation period and stable continuous extraction was
possible.
INDUSTRIAL APPLICABILITY
According to the processes of the present invention,
reduced coenzyme Q10 can be produced cheaply on the
industrial scale by considerably simple steps comprising
cuituring microorganisms and recovering reduced coenzyme
Q10. In addition, oxidized coenzyme Q10 can also be
produced by simple processes.

WE CLAIM:
1. A process for producing the oxidized coenzyme Q10 represented by the following
formula (I):

which comprises culturing reduced coenzyme Q10 producing microorganisms in a
culture medium containing a carbon source, a nitrogen source, a phosphorus source and a
micronutrient to obtain microbial cells containing reduced coenzyme Q10 at a ratio of not
less than 70 mole % among the entire coenzymes Q10
optionally disrupting the microbial cells; and
either oxidizing thus-produced reduced coenzyme Q10 to oxidized coenzyme Q10
using an oxidizing agent and then extracting the resultant by an organic solvent, or
extracting thus-producing reduced coenzyme Q10 by an organic solvent, purifying
optionally and oxidizing the resultant to oxidized coenzyme Q10 using an oxidizing agent.
2. The process as claimed in Claim 1,
wherein the production amount of reduced coenzyme Q10 on completion of the culture is
not less than 1 g/mL,
3. The process as claimed in Claim 1 or 2,
wherein the culture is carried out at 15 to 45°C and at a pH of 4 to 9.
4. The process as claimed in any one of Claims 1 to 3,
wherein the concentration of the carbon source in the culture is controlled to a
concentration that no substantially adverse effects are caused on the productivity of
reduced coenzyme Q10.
5. The process as claimed in Claim 4,

wherein the culture is carried out by a fed batch culture method.
6. The process as claimed in Claim 5,
wherein the carbon source is supplied to the culture medium separately from other
components.
7. The process as claimed in any one of Claims 1 to 6,
wherein the culture is carried out aerobically.
8. The process as claimed in any one of Claims 1 to 7,
wherein the microbial cells are disrupted in the extraction.
9. The process as claimed in Claim 8.
wherein the cell disruption is carried out by a physical treatment, a chemical treatment, an
enzymic treatment, a heating treatment, an autolysis, an osmolysis or a plasmoptysis.
10. The process as claimed in Claim 9,
wherein the cell disruption is carried out by a physical treatment.
11. The process as claimed in Claim 10,
wherein the physical treatment is carried out by a high pressure homogenizer, an
ultrasonic homogenizer, a French press or a ball mill.
12. The process as claimed in any one of Claims 1 to 11,
wherein the extraction of coenzymes Q10 is carried out from wet cells or dry cells of the
microbial cells or disrupted product thereof by using a hydrophilic organic solvent.
13. The process as claimed in any one of Claims 12,
wherein the hydrophilic organic solvent is acetone, aeetonitzile, methanol, ethanol, 1-
groganol or 2-propanol.
14. The process as claimed in any one of Claims 1 to 11,

wherein the extraction of the coenzymes Q10 is carried out from an aqueous suspension of
the microbial cells or disrupted product thereof by using a hydrophobic organic solvent.
15. The process as claimed in Claim 14,
wherein the hydrophobic organic solvent is a hydrocarbon, a fatty acid ester or an ether.
16. The process as claimed in Claim 14 or 15,
wherein the hydrophilic organic solvent is used as an auxiliary solvent in combination
with the hydrophobic organic solvent.
17. The process as claimed in Claim 16,
wherein the hydrophobic organic solvent is a hydrocarbon, and the hydrophilic organic
solvent is an alcohol.
18. The process as claimed in Claim 16,
wherein the hydrophobic organic solvent is an aliphatic hydrocarbon, and the hydrophilic
organic solvent is a nonohydric alcohol containing 1 to 5 carbon atoms.
19. The process as claimed in Claim 16,
wherein the hydrophobic organic solvent is at least one species of hexane and heptane,
and the hydrophilic organic solvent is at least one species of methanol, ethanol, 1-
propanol and 2-propanol.
20. The process as claimed in any one of Claims 16 to 19,
wherein the extraction is carried out under the condition that the hydrophobic organic
solvent is contained in 25 to 65% by volume and the hydrophilic organic solvent is
contained in 5 to 50% by volume.
21. The process as claimed in any one of Claims 16 to 20,
wherein the extraction is carried out by continuous extraction.
22. The process as claimed in any one of Claims 1 to 21,

wherein the reduced coenzyme Q10 is contained at a ratio of not less than 70 mole %
among the entire coenzymes Q10 wherein in the case that the reduced coenzyme Q10
producing microorganisms are cultured with shaking (amplitude: 2 cm, 310
reciprocation/min) at 25°C for 72 hours in 10 mL of a culture medium [(glucose: 20 g,
peptone: 5 g, yeast extract: 3g, malt extract: 3 g)/L, pH: 6.01 using a test tube (inner
diameter: 21 rnm, entire 35 length: 200 m),
the obtained broth is optionally concentrated,
the obtained solution is vigorously shaken for 3 minutes using 10 parts by volume
of glass beads (425 to 600 m) to disrupt the microorganisms under a nitrogen
atmosphere in the concomitant presence of 3 parts by volume of isopropanol and 18.5
parts by volume of n-hexane relative to 10 parts by volume of the broth, and
the prepared hydrophobic organic solvent phase (n-hexane phase) is analyzed by HPLC.
23. The process as claimed in any one of Claim 22,
wherein the reduced coenzyme Q10 producing microorganisms have not less than 1
pgimL of a productivity of reduced coenzyme Q10 per unit culture medium when
measured by HPLC under the condition as claimed in Claim 22.
24. The process as claimed in any one of Claims 1 to 23,
wherein the microorganisms are microorganisms of the genus Agrobacterium, the genus
Aspergillus, the genus Acetobacter, the genus Aminobacter, the genus Agromonas, the
genus Acidiphilium, the genus Bulleromyces, the genus Bullera, the genus
Brevundimonas, the genus Cryptococcus, the genus Chionosphaera, the genus Candida,
the genus Cerinosterus, the genus Exisophiala, the genus Exobasidium, the genus
Fellomyces, the genus Filobasidiella, the genus Filobasidium, the genus Geotrichum, the
genus Graphiola, the genus Gluconobacter, the genus Kockovaella, the genus
Kurtzmanomyces, the genus Lalaria, the genus Leucosporidium, the genus Legionella,
the genus Methylobacterlurn, the genus Mycoplana, the genus Oosporidium, the genus
Pseudomonas, the genus Psedozyma, the genus Paracoccus, the genus Petromyc, the
genus Rhodotorula, the genus Rhodos-oridium, the genus Rhizomonas, the genus
Rhodobium, the genus Rhodoplanes, the genus Rhodopseudomonas, the genus
Rhodobacter, the genus Sporobolomyces, the genus Sporidiobolus, the genus Saitoella,

the genus Schizosaccharomyces, the genus Sphingomonas, the genus Sporotrichum, the
genus Sympodiomycopsis, the genus Steriamatosporidium, the genus Tapharina, the
genus Tremella, the genus Trichosporon, the genus Tilletiaria, the genus Tilletia, the
genus Tolyposporium, the genus Tilletiopsis, the genus Ustilago, the genus Udeniomyce,
the genus Xanthophllomyces, the genus Xanthobacter, the genus Paecilomyces, the qenus
Acremonium, the genus Hyhomonus, or the genus Rhizobium.
25. The process as claimed in any one of Claims 1 to 24,
wherein the obtained oxidized coenzyme Q10 is purified optionally and crystallized to
obtain an oxidized
coenzyme Q10 crystal.

The present invention discloses a process for producing the oxidized coenzyme
Q10 which comprises culturing reduced coenzyme Q10 producing microorganisms in a
culture medium containing a carbon source, a nitrogen source, a phosphorus source
and a micronutrient to obtain microbial cells containing reduced coenzyme Q10 at a
ratio of not less than 70 mole % among the entire coenzymes Q10, optionally
disrupting the microbial cells; and either oxidizing thus-produced reduced coenzyme
Q10 to oxidized coenzyme Q10 using an oxidizing agent and then extracting the
resultant by an organic solvent, or extracting thus-producing reduced coenzyme Q10
by an organic solvent, purifying optionally and oxidizing the resultant to oxidized
coenzyme Q10 using an oxidizing agent.

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http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=4qWVaRRxoGSuNOShJ2/qUQ==&loc=wDBSZCsAt7zoiVrqcFJsRw==

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Patent Number

269183

Indian Patent Application Number

1902/KOLNP/2008

PG Journal Number

41/2015

Publication Date

09-Oct-2015

Grant Date

07-Oct-2015

Date of Filing

12-May-2008

Name of Patentee

KANEKA CORPORATION

Applicant Address

2-4, NAKANOSHIMA 3-CHOME, KITA-KU OSAKA-SHI, OSAKA

Inventors:

#	Inventor's Name	Inventor's Address
1	YAJIMA KAZUYOSHI	120-55-A804, KOKUBO, AKASHI-SHI, HYOGO 673-0005
2	KANDA AKIHISA	3-7-908, ASAHIMACHI, 3-CHOME, ABENO-KU, OSAKA-SHI, OSAKA, 545-0051
3	KITAMURA SHIRO	10-36-601, AIOICHO, 1-CHOME, AKASHI-SHI HYOGO 673-0882
4	KATO TAKAHISA	51-2-205, KOKUBO, AKASHI-SHI, HYOGO 673-0005

PCT International Classification Number

C12P 7/22, C12P 7/66

PCT International Application Number

PCT/JP2002/013766

PCT International Filing date

2002-12-27

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	2001-398545	2001-12-27	Japan