利瑪原甲藻岡田海綿酸的釋出與回收研究

Abstract

本研究試圖從利瑪原甲藻的培養液中回收其在生長中所釋出於培養基中的岡田海綿酸 (okadaic acid, OA)，並改良其提純方法，藉由細胞自然沉降濃縮與置換新鮮培養基的接種方式，提升利瑪原甲藻到前所未有的高細胞密度 (105 cells/mL以上)。在試圖以環境因子刺激OA釋放的研究中，發現間歇性手動搖晃與連續打氣刺激不會促進單位細胞釋毒率，且在隨後兩週的完全靜置情況下，細胞仍不斷釋出OA，其單位細胞釋毒率甚至比震盪處理下高，證明OA不若前人報告所稱需要攪動刺激而去酯反應釋出，在完全靜置情況下OA主動釋出到細胞外，而胞外高濃度的OA並不會抑制利瑪原甲藻的細胞生長，但降低其OA的釋放率，在低濃度胞外OA (0.51±0.03 mg/L)環境時，有較高的釋放率 (0.54±0.19 pg/cell/day)，而在高濃度胞外OA (3.74±0.2 mg/L) 環境時，其釋放率 (0.02±0.12 pg/cell/day) 大幅降低。高細胞密度培養下所釋出的OA極為可觀，利用合成吸附樹脂SP825充填的管柱，以幫浦設定不同流速輸送含OA的無細胞培養液通過管柱，測試SP825對OA的吸附效率，得知其吸附量至少有10 mg/g，隨後藉由甲醇的自動迴流萃取，脫附率可達97.35%；粗萃取物經過自動化速分管柱層析，提純率達90%以上。本研究探討培養中利瑪原甲藻細胞OA的釋出，並建立快速回收OA的方法，確定部分操作參數，以提供未來商業化生產的參考。

This study attempted to recover the released okadaic acid (OA) from the cultures of P. lima and improve the methodology of OA isolation. In P. lima culture, we found that OA was released and accumulated in the medium as mentioned elsewhere and in our previous studies. Thus, in order to enhance the OA production through the recovery from medium, high cell-density (over 105 cells/mL) cultures of P. lima were obtained by settling down the cells and replacing the old medium with fresh one. Treatments, such as increasing of vibration or aeration were found no enhancement in OA releasing rate of the cultured cells. It was found after a two-week period of static treatment of a culture in stationary phase, cells continuously released OA, and the releasing rate was even higher while comparing that with the vibration-treated culture at the same status. This result demonstrated that OA releasing in P. lima was spontaneous and was not as a result of stimulation from shaking or aerations reported elsewhere. It also showed that high concentration of extracellular OA did not affect the growth of P. lima cells, but inhibited the releasing of OA. In a medium of low extracellular OA content (0.51±0.03 mg/L), PL03 released OA at a higher rate (0.54±0.19 pg/cell/day) than that (0.02±0.12 pg/cell/day) of the cells maintained in a medium of higher extracellular OA (3.74±0.2 mg/L). The amount of OA released in high cell-density culture was substantially enormous. The revovery of OA from media was done by SP825 adsorption which was performed by a flow of medium through a column of 25 g SP825. Various flow rates had been tested for optimization and a capacity more than 10 mg OA/g SP825 was observed. The adsorbed toxin was easily eluted at a recovery of 97.35% by methanol using Soxlet reflux. The recovered crude extract was subjected for further CombiFlash column (Si) chromatographic separation to reach a purity more than 90% in the fractions collected. This study explored the releasing of OA in P. lima and established some parameters of OA recovery methodology for the future commercial production.