藉由微生物轉化生產橙皮素磷酸酯衍生物之研究

Abstract

橙皮素(hesperetin)為類黃酮化合物，常見於柑橘類果實中，屬於植物次級代謝物，具有預防心血管疾病、提升免疫力、抗氧化等生理活性。然而橙皮素的水溶性不佳，生物可利用率(bioavailability)極低，使其在應用上受到許多限制。本研究室先前篩選出Bacillus subtilis BCRC 80517菌株中具有類黃酮磷酸酯合成酶(flavonoid phosphate synthetase) 可將類黃酮磷酸酯化以增加其水溶性，進而增加其生物可利用率，本研究以此為基礎，利用5-L發酵槽進行橙皮素生物轉換的探討及建立下游回收橙皮素磷酸酯最適之程序。 實驗分為兩部分，第一部分為利用5 L通氣攪拌式發酵槽生產hesperetin 7-O-phosphate (H7P) 與hesperetin 3’-O-phosphate (H3’P)，合稱為HPs，結果顯示，最佳化生產條件為接種10%種菌，菌體生長至OD600 = 1.0批次饋入hesperetin 20 g/L，於37 oC、pH 7.0、450 rpm、1 vvm (air)、0.02 (s-1) 體積氧氣質傳係數 (k¬La)，並以氨水調控pH，可於發酵時間31 h，達到81%的轉換率，生成橙皮素磷酸酯20.3 g/L。第二部分為建立發酵液回收HPs之最適化程序。結果顯示，發酵液經過高速離心去除菌體，將上清液濃縮至HPs 200 mM，滴入6.7 M之CaCl2(aq)，使 hesperetin phosphate calcium salt (HP-Ca2+) 沉澱，得到HP-Ca2+之純度及回收率為53% (w/w)與85%。此外欲增加HP-Ca2+純度，將HP-Ca2+粉末懸浮於1:10 (w/v) 40%酒精攪拌洗滌2小時，得到HP-Ca2+之純度及回收率為58% (w/w)與90%。最後，為了使HP-Ca2+回溶於水後完全澄清，將酒精洗滌烘乾後之HP-Ca2+粉末回溶於水，過濾去除不溶物，取上清液進行凍乾，最終產物HP-Ca2+之純度為70.0% (w/w)。

Hesperetin (Hes), a flavanone with numerous physiological properties such as reducing cardiovascular disease and antioxidant, is the predominant flavonoid in lemons and oranges. However, the application of Hes is restricted by its low water solubility and poor bioavailability. In our previous studies, we found a unique enzyme, namely flavonoid phosphate synthetase, from B. subtilis BCRC 80517, could phosphorylate hesperetin to generate two highly water-soluble hesperetin phosphate derivatives. This study aimed to produce two hesperetin phosphate derivatives (HPs) through a 5-L aerated stirred-tank bioreactor and develop a feasible process for HPs recovery from fermentation broth. The results showed that the optimal condition for biotransformation process was that inoculated 10% of the seed culture, and feeding hesperetin (20 g/L) at OD600 1.0. The biotransformation process was performed at 37oC, pH 7.0, 450 rpm, 1.0 vvm (air), 0.02 (s-1) volume oxygen mass transfer coefficient (kLa) for 31 h. The main products were hesperetin 7-O-phosphate (H7P) and hesperetin 3'-O-phosphate (H3'P). The total content of HPs in the harvested fermentation broth was 20.3 g/L, and the conversion rate was 81%. The process for HPs recovery was described as follows. First of all, the harvested broth was centrifuged and then passed through a microfiltration system to remove the biomass, the permeate was concentrated to 200 mM of HPs and precipitated by adding CaCl2(aq), to obtain the HP-Ca2+ crude products. The crude products was washed with 40% (w/v) EtOH and then resuspended in water to remove the inpurities. Consequently, the supernatant was lyophilized to obtain HP-Ca2+ powder as the final product with around 70% (w/w) purity.