JP2021100437A - アスタキサンチン製造のためのヘマトコッカスの培養方法 - Google Patents
アスタキサンチン製造のためのヘマトコッカスの培養方法 Download PDFInfo
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- astaxanthin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
- C12N2529/10—Stimulation by light
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Abstract
Description
基材を用意する工程、
前記基材表面上にヘマトコッカスを配置する工程、及び
前記基材上に配置されたヘマトコッカスを培養プロセスの開始から高光強度に暴露する工程、ただし
前記ヘマトコッカスを低光エネルギーに暴露して初期培養を行う第1段階と、その後に前記第1段階で適用されたものよりも高い光エネルギーに前記ヘマトコッカスを暴露してアスタキサンチン合成を誘導することにより前記ヘマトコッカスを引き続き培養する第2段階とを含むヘマトコッカスの2段階培養プロセスを行わないこと、
を含み、さらに
培養されたヘマトコッカスを回収する工程、及び/又は
前記アスタキサンチンを単離する工程、
を所望により含む、アスタキサンチン製造のためのヘマトコッカスの培養方法によって解決される。
実験設計
ベンチスケールのバイオフィルムフォトバイオリアクター
使用されたベンチスケールの二層フォトバイオリアクター(PBR)は、Schultze et al.(2015)(参照によって援用される)に記載されたものであった。概要としては、この系は、ポリ塩化ビニル(PVC)製支持体上の透明なPMMA管(長さ50cm、直径12cm)内に垂直に設置されたグラスファイバーマット(50×10cm)からなる。培地が蠕動ポンプ(peristaltic pump)によって一定に循環されている。培地はグラスファイバーの頂部から付与され、重力により下に拡散し、PVC支持体内に設置された培地貯留槽に戻る。この系には1Lの培地が供給され、藻類の成長用の栄養が制限されることを防ぐため2〜3日毎に交換される。通気はPVC管内に供給される。
各サンプリング時点において、少なくとも3枚のフィルターを各PBRから採取した。播種領域を超えて増殖したバイオマスを除去し、フィルターを凍結乾燥して恒量とした。乾燥重量を重量測定法で求め、アスタキサンチン解析までバイオマスを−20℃で保存した。
アスタキサンチンをLi et al.(2012)に記載されるように分光測定した。凍結乾燥されたバイオマス試料をジメチルスルホキシド(DMSO、メルク社、ダルムシュタット、ドイツ)で抽出し、70℃で5分間インキュベートし、次いで4000Gで5分間遠心した。無色のペレットが得られるまで抽出を繰り返した。上清を集め、530nmでODを測定した(Infinite M200プレートリーダー、テカン社(Tecan)、メンネドルフ、スイス)。完全な抽出に必要な場合、砂ですりつぶして細胞を破砕した。DMSOに溶解及び希釈したアスタキサンチン標準物質(純度98.6%、ドクトル・エレンストルファー社(Dr. Ehrenstorfer)、アウクスブルク、ドイツ)を用いて作成した検量線に基づいて、アスタキサンチン濃度を決定した。
Aflalo C, Meshulam Y, Zarka A, Boussiba S (2007) On the Relative Efficiency of Two- vs. One-stage Production of Astaxanthin by the Green Alga Haematococcus pluvialis. Biotechnol Bioeng 98: 300-305. doi: 10.1002/bit.21391
Berner F, Heimann K, Sheehan M (2014) Microalgal biofilms for biomass production. J Appl Phycol. doi: 10.1007/s10811-014-0489-x
Del Rio E, Acien FG, Garcia-Malea MC, et al (2005) Efficient one-step production of astaxanthin by the microalga Haematococcus pluvialis in continuous culture. Biotechnol Bioeng 91: 808-815. doi: 10.1002/bit.20547
Dong, S., Huang, Y., Zhang, R., Wang, S., & Liu, Y. (2014). Four Different Methods Comparison for Extraction of Astaxanthin from Green Alga Haematococcus pluvialis, 2014
Fiedler, D., Hager, U., Franke, H., Soltmann, U., Bottcher, H., (2007) Algae biocers: astaxanthin formation in sol-gel immobilised living microalgae. J. Mater. Chem. 2007, 17, 261-266
Garcia-Malea LopezMC, Sanchez EDR, Casas Lopez JL, et al (2006) Comparative analysis of the outdoor culture of Haematococcus pluvialis in tubular and bubble column photobioreactors. J Biotechnol 123: 329-342. doi: 10.1016/j.jbiotec.2005.11.010
Li Y, Miao F, Geng Y, et al (2012) Accurate quantification of astaxanthin from Haematococcus crude extract spectrophotometrically. Chinese J Oceanol Limnol 30: 627-637. doi: 10.1007/s00343-012-1217-5
Liu T, Wang J, Hu Q, et al (2013) Attached cultivation technology of microalgae for efficient biomass feedstock production. Bioresour Technol 127: 216-222. doi: 10.1016/j.biortech.2012.09.100
Lorenz RT, Cysewski GR (2000) Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol 18: 160- 167.
Murphy T, Fleming E, Bebout L, et al (2012) A Novel Microbial Cell Cultivation Platform for Space Applications. In: 1st Annual International Space Station Research and Develop- ment Conference. Denver, CO: American Astronomical Society (AAS). pp 335-339
Naumann T, Cebi Z, Podola B, Melkonian M (2013) Growing microalgae as aquaculture feeds on twin-layers: a novel solid-state photobioreactor. J Appl Phycol 25: 1413-1420. doi: 10.1007/s10811-012-9962-6
Nguyen K (2013) Astaxanthin: a comparative case of synthetic vs. natural production. Chem Biomol Eng Publ Other Work 1: 1-11.
Nobre, B., Marcelo, F., Passos, R., Beirao, L, Palavra, A., Gouveia, L, & Mendes, R. (2006). Supercritical carbon dioxide extraction of astaxanthin and other carotenoids from the microalga Haematococcus pluvialis. European Food Research and Technology, 223, 787-790. Retrieved from http://dx.doi .org/10.1007/s00217-006-0270-8
Nowack ECM, Podola B, Melkonian M (2005) The 96-well Twin-Layer system: A novel approach in the cultivation of microalgae. Protist 156: 239-251. doi: 10.1016/j.protis.2005.04.003
Olivieri G, Salatino P, Marzocchella A (2014) Advances in photobioreactors for intensive microalgal production: configurations, operating strategies and applications. J Chem Technol Biotechnol 89: 178-195. doi: 10.1002/jctb.4218
Ozkan A, Kinney K, Katz L, Berberogiu H (2012) Reduction of water and energy requirement of algae cultivation using an algae biofilm photobioreactor. Bioresour Technol 114: 542-8. doi: 10.1016/j.biortech.2012.03.055
Schultze LKP, Simon M-V, Li T, et al (2015) High light and carbon dioxide optimize surface productivity in a Twin-Layer biofilm photobioreactor. Algal Res 8C: 37-44. doi: 10.1016/j. algal.2015.01.007
Suh IS, Joo H-N, Lee C-G (2006) A novel double-layered photobioreactor for simultaneous Haematococcus pluvialis cell growth and astaxanthin accumulation. J Biotechnol 125: 540-6. doi: 10.1016/j.jbiotec.2006.03.027
Wan M, Hou D, Li Y, et al (2014) The effective photoinduction of Haematococcus pluvialis for accumulating astaxanthin with attached cultivation. Bioresour Technol 163C: 26-32. doi: 10.1016/j. biortech.2014.04.017
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Claims (7)
- 基材を用意する工程、
前記基材表面上にヘマトコッカスを配置する工程、並びに
前記ヘマトコッカスを低光エネルギーに暴露して初期培養を行う第1段階と、その後に前記第1段階で適用されたものよりも高い光エネルギーに前記ヘマトコッカスを暴露してアスタキサンチン形成を誘導することにより前記ヘマトコッカスを引き続き培養する第2段階とを含むヘマトコッカスの2段階培養プロセスを行わずに、前記基材上に配置された前記ヘマトコッカスを培養プロセスの開始から高光強度に暴露する工程、
を含み、さらに
培養された前記ヘマトコッカスを回収する工程、及び/又は
アスタキサンチンを単離する工程、
を所望により含む、アスタキサンチン製造のためのヘマトコッカスの培養方法。 - 回収されたヘマトコッカスから前記アスタキサンチンが単離される、請求項1に記載の方法。
- 前記ヘマトコッカスがヘマトコッカス・プルビアリス(Haematococcus pluvialis)である、請求項1又は請求項2に記載の方法。
- 前記第1段階における前記低光エネルギーが約50μmol photons m−2s−1以上約200μmol photons m−2s−1未満である、請求項1〜請求項3のいずれか1項に記載の方法。
- 前記第2段階における前記高い光エネルギーが約200μmol photons m−2s−1以上であり、特に約500μmol photons m−2s−1以上である、請求項1〜請求項4のいずれか1項に記載の方法。
- 前記基材がシート状の材料であり、特に多孔質シートである、請求項1〜請求項5のいずれか1項に記載の方法。
- 前記シート状の材料が、紙、セルロースエステル、特に酢酸セルロース、セルロース混合エステル、セルロース、硝酸セルロース、ポリアミド、ポリエステル及び/又はポリオレフィンからなる群から選択される、請求項6のいずれか1項に記載の方法。
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