Process for extraction of carotenoids from bacterial cells

Abstract: A process for extraction of a carotenoid compound from bacterial cells containing the carotenoid compound comprising the step of bringing the bacterial cells into contact with supercritical fluid so as to extract the carotenoid compound from the cells. The process provides carotenoid compounds which can be safely used as feed additives and food additives.

Description: BACKGROUND OF THE INVENTION 1. Field of Invention The present invention relates to a process for extraction of a carotenoid compound from bacterial cells containing the carotenoid compound. 2. Related Art Carotenoid compounds are red-yellow series pigments widely distributed in nature, such as in microbial cells, algal cells, and organs and tissues of plants and animals. The carotenoid compounds are used in the field of food additives as coloring agents for foods and beverages, and feed additives for coloring meat or skin of fish such as salmon and trout, and for coloring eggs of poultry such as chicken eggs; recently, the use of carotenoid compounds has been expanding. The carotenoid compounds are known to have anti-oxidant action, and are expected to be useful as anti-oxidant agents. In addition, it was recently found that some carotenoid compounds have anti-cancer effect, and therefore are expected to be useful as medicaments. The carotenoid compounds are classified into those containing an oxygen atom in the molecule and those containing no oxygen atom, and the former are called xanthophyll compounds. Among the xanthophyll compounds, astaxanthin, canthaxanthin, zeaxanthin etc. are industrially produced by chemical synthesis (Pure and Applied Chemistry, 63 (1), 35-44 (1991)), and are used as feed additives for coloring. However, recently it has become difficult to use chemically synthesized product as food or feed additive due to safety issues, and therefore the development of a process for production of natural carotenoid compounds replacing the chemically synthesized compounds is urgently sought. For industrial production of carotenoid compounds it is considered that the best process is that comprising culturing bacterial cells to accumulate a carotenoid, recovering the cultured bacterial cells, and extracting the carotenoid compound from the cells, considering stable supply and high growth rate and productivity. Japanese Unexamined Patent Publication (Kokai) Nos. 2-138996, 6-165683 and 6-165684 describe methods for extraction of carotenoid compounds from bacterial cells, all of which methods use organic solvents for the extraction. However, since the extracted carotenoid compounds are used as food additives or feed additives, residual solvent may cause problems. Therefore the selection of solvents which can be used for extraction is limited. In addition, xanthophyll compounds which are a group of carotenoid compounds hardly dissolve in solvents which are considered to be safe. Biotechnal Prog.1990, 6, pages 82-91, M.L.Cygnarowicz and W.D.Seider describes measuring the solubility of pure carotene in supercritical CO 2 alone and with 1 wt.% of methanol or ethanol or methylene chloride, ethanol giving the greatest increase in solubility. It also discloses a theoretical design (with theoretical results) for extracting β-carotene from crushed cells obtained from fermentation broths, the extraction using supercritical CO 2 alone or in combination with 1 wt.% ethanol. JP-A-06200179 describes extracting astaxanthin from powdered krill shell using supercritical CO 2 . JP-A-60035057 describes obtaining crude astaxanthin from Euphasia superba by extraction with organic solvent, treating the crude to neutralise it and remove lipids, and extracting astaxanthin from the reaction mixture with supercritical liquid such as CO 2 . SUMMARY OF THE INVENTION The present invention provides a process for extraction of a carotenoid compound from bacterial cells containing the carotenoid compound comprising contacting the bacterial cells with a supercritical fluid in combination with entrainer selected from methanol and isopropanol so as to extract the carotenoid compound from the bacterial cells, the bacterial cells being wet cells, dried cells or cells suspended in a solvent. A supercritical fluid is a fluid having a distribution constant similar to that of gases, a specific gravity near to that of liquids and a solubilizing power similar to that of liquids. DETAILED DESCRIPTION As a supercritical fluid used in the present invention, supercritical carbon dioxide is preferred. The supercritical carbon dioxide is carbon dioxide having a temperature the same as or higher than the critical temperature of 31.1°C and a pressure the same as or higher than the critical pressure of 7375 kPa (75.2 kgf/cm 2 ). The extraction may be carried out in a pressure-resistant container. The amount of the entrainer is usually at most 100% by weight relative to the supercritical fluid, though the amount is not limited thereto. The entrainer is selected from methanol and isopropylalcohol (IPA). Any extraction temperature the same as or higher than the critical temperature of the supercritical fluid used can be used. If supercritical carbon dioxide is used, the extraction temperature is the same as or higher than the critical temperature of carbon dioxide (31.1°C) - usually up to 100°C, more preferably up to 60°C, and even more preferably up to 45°C. Any extraction pressure the same as or higher than the critical pressure of the supercritical fluid used can be used. If supercritical carbon dioxide is used, the extraction pressure is the same as or higher than the critical pressure of carbon dioxide - 7375 kPa (75.2 kgf/cm 2 ) - and usually up to 49 mPa (500 kgf/cm 2 ), preferably 24.5 mPa (250 kgf/mm 2 ). The extraction time is not critical, but is preferably at least one second - (more preferably at least one minute, and most preferably at least 10 minutes) and up to 100 hours (more preferably up to 10 hours). The amount of supercritical fluid used is not critical, and is usually at least 1 mg, e.g. 1 mg to 100g, per 1 mg of bacterial cells; preferably it is at least 10 mg, and up to 10g and more preferably up to 5g, per 1 mg of bacterial cells. The materials to be subjected to the present extraction may be any bacterial cells containing a carotenoid compound. Examples of such bacterial cells are those obtained by culturing a bacterium having an ability to produce at least one carotenoid compound selected from the group consisting of astaxanthin, adonixanthin, β-carotene, echinenone, canthaxanthin and zeaxanthin, such as the E-396 strain (FERM BP-4283) in a medium containing a carbon source, nitrogen source, inorganic salts and if necessary other required substance (for example, vitamins, amino acids, nucleotides etc.), and centrifuging or filtering the culture or by other conventional methods. Alternatively, the present cells containing a carotenoid can be obtained by culturing a bacterium belonging to the genus Corynebacterium having an ability to produce canthaxanthin, such as Corynebacterium SQH348 (FERM BP-4284) in a medium containing carbon source, nitrogen source, inorganic salts and if necessary other required substances (for example, vitamins, amino acids and nucleotides), and recovering the cells by centrifugation or filtration. Again, the bacterial cells of the present invention are not limited to those exemplified above. Bacterial cells to be subjected to extraction may be wet cells, dried cells, or cells suspended in a solvent. The carotenoid compounds to be extracted are any carotenoid compounds. The present invention is especially useful for extraction of the xanthophyll compounds which are not usually extracted by any conventional process. The carotenoids extracted according to the present invention are, for example, astaxanthin, canthaxanthin, zeaxanthin, adonixanthin, echinenone, cryptoxanthin, adonirubin, asteroidenon, rhodoxanthin, 3-hydroxyechinenon, astaxanthin ester, β-carotene, α-carotene, γ-carotene, lycopene, capsanthin, β-zeacarotene, torulene, flanoxanthin, α-doracarotene, fucoxanthin and phoenicoxanthin. According to the present invention, bacterial cells containing at least one of the above-mentioned carotenoid compounds can be used. Especially, bacterial cells containing at least one of astaxanthin, canthaxanthin, zeaxanthin, adonixanthin, echinenone, cryptoxanthin, adonirubin and asteroidenone are preferably used. EXAMPLES The present invention is further explained by the following Examples, of which Examples 10-17, 30-37, 42, 44, 47, 48, 52 and 53 are according to the invention with the remainder being for comparison or to show cell preparation. Preparative Example 1 An astaxanthin and adonixanthin-producing bacterium E-396 (FERM'BP-4283) was cultured in 30L of a medium containing 20 g/L yeast extract, 30 g/L sucrose, 1.5 g/L KH 2 PO 4 , 1.5 g/L Na 2 HPO 4 , 0.5 g/L MgSO 4 •7H 2 O, 0.01 g/L FeSO 4 •7H 2 O, 0.01 g/L CaCl 2 , adjusted to pH 7.0 with Na 2 CO 3 for 5 days, and the cultured cells were centrifuged to obtain 900g of wet cells. The wet cells contained 1.0 mg/g of carotenoid compounds. 300g of the wet cells thus obtained was lyophilized to obtain 57g of lyophilized cells. Examples 1 to 20 50 mg of the dried cells of E-396 (FERM BP-4283) was added to a HPLC column having a capacity of 2.5 ml, and the column was connected to a supercritical chromatography apparatus (Nippon Denshi K.K.). Extraction was carried out by passing supercritical carbon dioxide through the column at a flow rate of 92 mg/min. If an entrainer was used, extraction was carried out by flowing the supercritical carbon dioxide at a flow rate of 92 mg/min. and the entrainer at a flow rate of 20 µL/min. The extraction temperature, extraction pressure and entrainer conditions were varied as shown in Table 1, and the results shown in Table 1 were obtained. The extraction ratio is the ratio of the amount of extracted carotenoid to the amount of carotenoid contained in the cells prior to the extraction. Example Extraction temperature °C Extraction pressure mPa (kgf/cm 2 ) Extraction time min Entrainer Extraction ratio % 1 35 19.6 (200) 30 No 42 2 35 19.6 (200) 60 No 63 3 35 19.6 (200) 120 No 75 4 35 19.6 (200) 240 No 92 5 40 19.6 (200) 60 No 55 6 50 19.6 (200) 120 No 38 7 35 24.5 (250) 60 No 64 8 35 14.7 (150) 60 No 59 9 35 9.8 (100) 60 No 41 10 35 9.8 (100) 60 Methanol 70 11 35 14.7 (150) 60 Methanol 75 12 35 19.6 (200) 60 Methanol 79 13 35 9.8 (100) 120 Methanol 93 14 35 9.8 (100) 60 IPA 95 15 35 14.7 (150) 60 IPA 93 16 35 19.6 (200) 60 IPA 93 17 35 9.8 (100) 120 IPA 99 18 35 9.8 (100) 60 Acetone 64 19 35 14.7 (150) 60 Acetone 67 20 35 19.6 (200) 60 Acetone 66 Examples 21 to 40 100 mg of the wet cells of E-396 (FERM BP-4283) was spread on a rectangular mesh, and the mesh was added to a HPLC column having a capacity of 2.5 mL. The column was connected to a supercritical chromatography apparatus, and extraction was carried out under the same conditions as described above. The extraction temperature, extraction pressure and entrainer conditions were varied as shown in Table 2, and the results are shown in Table 2. The extraction ratio is the ratio of the amount of extracted carotenoid to the amount of carotenoid contained in the cells prior to the extraction. Example Extraction temperature °C Extraction pressure mPa (kgf/cm 2 ) Extraction time min Entrainer Extraction ratio % 21 35 19.6 (200) 30 No 42 22 35 19.6 (200) 60 No 63 23 35 19.6 (200) 120 No 75 24 35 19.6 (200) 240 No 92 25 40 19.6 (200) 60 No 55 26 50 19.6 (200) 120 No 38 27 35 24.5 (250) 60 No 64 28 35 14.7 (150) 60 No 59 29 35 9.8 (100) 60 No 41 30 35 9.8 (100) 60 Methanol 70 31 35 14.7 (150) 60 Methanol 75 32 35 19.6 (200) 60 Methanol 79 33 35 9.8 (100) 120 Methanol 93 34 35 9.8 (100) 60 IPA 95 35 35 14.7 (150) 60 IPA 93 36 35 19.6 (200) 60 IPA 93 37 35 9.8 (100) 120 IPA 99 38 35 9.8 (100) 60 Acetone 64 39 35 14.7 (150) 60 Acetone 67 40 35 19.6 (200) 60 Acetone 66 Example 41 10g of the dried cells of E-396(FERM BP-4283) was put into a 100 mL-high pressure container, and supercritical carbon dioxide at 35°C and 14.7 mPa (150 kgf/cm 2 ) was passed through the container at a flow rate of 10 g/min. for 2 hours to extract carotenoid compounds. As a result, the extraction ratio was 75%. Example 42 10g of the dried cells of E-396 (FERM BP-4283) was mixed with 20 ml of isopropanol and the mixture was added to a 100 ml high pressure container. Supercritical carbon dioxide at 35°C and 14.7 mPa (150 kgf/cm 2 ) was passed through the container at a flow rate of 10 g/min. for 2 hours to extract carotenoid compounds. As a result, the extraction ratio of the carotenoid compound was 97%. Example 43 20g of the wet cells of E-396 (FERM BP-4283) was added to a 100 ml high pressure container, and supercritical carbon dioxide at 35°C and 14.7 mPa (150 kgf/cm 2 ) was passed through the container at a flow rate of 10 g/min. for 2 hours to extract carotenoid compounds. As a result, the extraction ratio of the carotenoid compound was 64%. Example 44 20g of the wet cells of E-396 (FERM BP-4283) was mixed with 20 mL of isopropanol, and the mixture was added to a 100 mL high pressure container. Supercritical carbon dioxide at 35°C and 14.7 mPa (150 kgf/cm 2 ) was passed through the container at a flow rate of 10 g/min. for 2 hours to extract carotenoid compounds. As a result, the extraction ratio of the carotenoid compound was 95%. Preparative Example 2 A canthaxanthin-producing strain, Corynebacterium sp. SQH348 (FERM BP-4284), was cultured in 3L of a medium containing 30 g/L yeast extract, 10 g/L glucose, 5 ml/L soy bean oil, 2.5 g/L NH 4 NO 3 , 1.5 g/L KH 2 PO 4 , 1.5 g/L Na 2 HPO 4 , 0.5 g/L MgSO 4 •7H 2 O, 0.01 g/L FeSO 4 •7H 2 O and 0.01 g/L CaCl 2 , adjusted to pH 8.0 with Na 2 CO 3 for 7 days, and the culture was centrifuged to obtain 75g of the wet cells. The wet cells contained 0.61 mg/g carotenoid compounds. 40g of the wet cells was lyophilized to obtain 8.6g of lyophilized cells. Examples 45 to 49 50 mg of the dried cells of Corynebacterium sp. SQH348 (FERM BP-4284) was filled in a HPLC column having a capacity of 2.5 mL, and the column was connected to a supercritical chromatography apparatus (Nippon Denshi K.K.). Extraction was carried out by passing supercritical carbon dioxide through the column at a flow rate of 92 mg/min. If an entrainer was used, the supercritical carbon dioxide was flowed at a flow rate of 92 mg/min. and the entrainer was flowed at a flow rate of 20 µL/min. The results are shown in Table 3. Examples 50 to 54 100 mg of the wet cells of Corynebacterium sp. SQH348 (FERM BP-4284) was spread on a rectangular mesh, which was then added to a HPLC column having a capacity of 2.5 mL. The column was joined to a supercritical chromatography apparatus. Extraction was carried out under the same conditions as described in Examples 45 to 49. The results are shown in Table 3. Example Extraction temperature °C Extraction pressure mPa (kgf/cm 2 ) Extraction time min Entrainer Extraction ratio % 45 35 19.6 (200) 120 No 78 46 35 9.8 (100) 120 No 66 47 35 9.8 (100) 60 Methanol 74 48 35 9.8 (100) 60 IPA 94 49 35 9.8 (100) 60 Acetone 68 50 35 19.6 (200) 120 No 72 51 35 9.8 (100) 120 No 59 52 35 9.8 (100) 60 Methanol 71 53 35 9.8 (100) 60 IPA 90 54 35 9.8 (100) 60 Acetone 65 According to the present invention, carotenoid compounds, especially xanthophyll compounds, can be efficiently extracted from bacterial cells. Carotenoid compounds produced by the present invention can be safely used as feed additives and food additives.

Claims: A process for extraction of a carotenoid compound from bacterial cells containing the carotenoid compound comprising contacting the bacterial cells with a supercritical fluid in combination with entrainer selected from methanol and isopropanol so as to extract the carotenoid compound from the bacterial cells, the bacterial cells being wet cells, dried cells or cells suspended in a solvent. A process according to claim 1, wherein the carotenoid compound is a xanthophyll compound. A process according to claim 1 or 2 wherein the supercritical fluid is supercritical carbon dioxide. A process according to any preceding claim wherein the amount of the supercritical fluid used is between 1 mg and 100 g per 1 mg bacterial cells. A process according to any preceding claim wherein the entrainer is used in an amount of up to 100% by weight relative to the amount of supercritical fluid.

Inventors: KITAOKA MOTOMITSU, KIYOTA TAKASHI, TSUBOKURA AKIRA, KITAOKA, MOTOMITSU, KIYOTA, TAKASHI, TSUBOKURA, AKIRA, Kitaoka, Motomitsu, c/o Central Technical Research, Kiyota, Takashi, c/o Central Technical Research, Tsubokura, Akira, c/o Central Technical Research
Applicants: NIPPON MITSUBISHI OIL CORP, NIPPON MITSUBISHI OIL CORPORATION
Application year: 1995
Main classification: C12P23
IPC codes: C07C403, B01D11, C12P23, C12R1