利用PCS生物反應器以Rhizopus oligosporus NTU 5進行黑豆漿發酵並探討異黃酮去醣基化效率及生理活性之研究

Abstract

近年來，許多研究結果顯示去醣基大豆異黄酮之生理活性及吸收效果皆强於帶醣基之大豆異黄酮。因此，學者們致力於探討如何利用固態發酵進行大規模的大豆異黃酮之生物轉化。 然而，費時及難以量化生產是其所面臨的難題。 Plastic composite support (PCS), 是一種利用聚丙烯經高溫擠壓將營養物質包覆其中的载體。過去研究顯示，利用PCS作為固定化載體於重複批次及連續發酵中， 確實可以增加發酵產物產量。 本研究之目的主要是評估利用PCS讓根黴菌Rhizopus oligosporus NTU 5附著後進行黑豆漿發酵之效果， 並探討在發酵過程中pH之控制與否對黑豆漿的異黃酮去醣基化效率之影響， 以及分析發酵黑豆漿之β-glucosidase活性、清除自由基能力、總多酚含量及抗發炎能力。在進行生物反應器發酵前，本實驗探討了4種具有不同營養成分的PCS其讓根黴菌附著的能力。這4種PCS分別命名為S, SF+, SFYB+, 和SFYBR，其英文字母符號代表其PCS所含有的營養成分：S代表大豆殼粉、F代表脫脂大豆粉、Y代表酵母提取物、B代表蛋白腖、R代表豬紅血球粉及+代表礦物鹽 ）。 結果顯示， 在四種PCS當中，S明顯地具有最多菌絲附著（0.237 ± 0.022 g）。因此，S被選用作為固定在生物反應器的PCS。本實驗研究所使用的黑豆漿含有大約190 mg/ L 的異黃酮總量. 研究結果顯示， 相較於控制pH於pH 6.5， 發酵過程中不控制pH具有比較好的異黃酮去醣基化效率。此外，利用PCS讓根黴菌菌絲附著不但可以持續釋放出β-glucosidase，提高異黃酮去醣基化效率，並將其去醣基化所需的時間縮短，可在8小時完成，其穩定性可達34天並完成了26批黑豆漿發酵。發酵黑豆漿的總多酚含量比未發酵黑豆漿多（0.042 ± 0.015 mg GAE/ mL sample），而不控制pH發酵黑豆漿的總多酚含量（範圍從 0.147 到 0.340 mg GAE/ mL sample） 比控制pH發酵黑豆漿的總多酚含量多（範圍從0.121 到 0.151 mg GAE/ mL sample）。然而，第22批發酵了8小時的豆漿具有最高的DPPH清除自由基能力（54.66 ± 0.39%）, 但卻不是擁有最多的總多酚含量（0.249 ± 0.002 mg GAE/ mL sample）。至於抗發炎能力分析，發酵黑豆漿粗萃取物無法抑制發炎因子iNOS 和COX-2，需做進一步之分離純化分析。

Numerous studies have revealed that isoflavones in their aglycone forms exhibit higher biological activity and are metabolically active. Isoflavone conversion of soybeans has been conducted using solid-state fermentation for years. However, the limitations are time consuming and the difficulty in scaling up for food industry. Plastic composite support (PCS), an extrusion product of a mixture between polypropylene and nutritious compounds, has been used to enhance various products in repeated-batch and continuous fermentation. The goal of this study is to evaluate the effects of PCS implementation on isoflavone deglycosylation in black soymilk fermented by Rhizopus oligosporus NTU 5, which is divided into two specific objectives: to evaluate the isoflavone deglycosylation of fermented black soymilk with and without pH control in PCS bioreactors, and to analyze the β-glucosidase activity, total phenolic content, antioxidant and anti-inflammatory activities in fermented black soymilk. Prior to bioreactor fermentation, optimal PCS for mycelia immobilization was selected by test tube cultivation. Four types of PCS were using namely S, SF+, SFYB+, and SFYBR, in which alphabetic character on behalf of the nutrients contained in PCS: S represents dried ground soybean hulls, F represents defatted soybean flour, Y represents yeast extract ardamine, B represents peptone, R represents dried porcine red blood cell, and + represents mineral salts. Significant results showed that S possessed the most mycelium weight (0.237 ± 0.022 g, p < 0.05) among the PCS studied, therefore, was selected for further study. In this study, non-fermented black soymilk contained proximately 190 mg/ L of total isoflavone content. It was found that the non pH control fermentation has better efficiency of isoflavone bioconversion when compared with the one with pH control at pH 6.5. Thus, non pH control fermentation was further studied for its long run fermentation efficiency. PCS bioreactor indeed enhances the isoflavone bioconversion process by continuously releasing β-glucosidase, which can be completed between 8 to 36 h. PCS bioreactor also provides a long run repeated batch fermentation which can be sustained for 34 days for 26 batches of black soymilk fermentation. A higher total phenolic content was found in fermented BSM from both fermentation conditions than non-fermented BSM (0.042 ± 0.015 mg gallic acid equivalents (GAE)/ mL) in present study. Obviously, the non pH control fermented BSM (range from 0.147 to 0.340 mg GAE/ mL sample) had higher total phenolic content compared to the one with pH control fermentation (range from 0.121 to 0.151 mg GAE/ mL sample). Even more remarkable was the black soymilk from 22nd batch with 8 h fermentation has the highest DPPH radical scavenging effect (54.66 ± 0.39%), yet its total phenolic content was 0.249 ± 0.002 mg GAE/ mL sample, which did not contain the highest total phenolic content among the samples studied. None of black soymilk sample fermented by R. oligosporus possessed with inhibitory effect that could suppress the protein expression of inflammatory genes inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Purification and fractionation of fermented black soymilk crude extract merit for further cell-based investigation.