加工後剩餘的可可果莢(CPH)約佔果實重量的74%，研究指出CPH中含有豐富的酚類化合物，是具有抗氧化活性之重要成分。超臨界CO2萃取技術具有低操作溫度、高擴散能力、無污染等多項優點，為萃取高價值天然產物的首選之一。酚類化合物多以酯鍵鍵結於植物細胞壁上，為提高植物細胞中酚類化合物的釋放，本研究以酵素作用輔助超臨界CO2萃取，建立CPH酚類化合物萃取方法。實驗分成兩階段進行，第一階段將CPH以熱風乾燥並磨粉過篩，使用四因子三階層之Box-Behnken反應曲面法，進行超臨界CO2萃取條件最適化，根據實驗數據建立二次多項方程式，以61℃、4735 psi及共溶劑比例17.4%萃取118分鐘為最佳萃取條件。第二階段將新鮮CPH泥分別以RAPIDASE®FIBER (R-F)、RAPIDASE®TF、BREWERS COMPASSTM和VALIDASE®TRL四種酵素，於不同酵素濃度(0.5-3.0%)和反應時間(0-6 hr)在50℃、pH 5.5進行水解，探討其還原醣及總酚含量的變化，選出最適酵素水解條件；最後將二階段合併，CPH經酵素水解後乾燥，以最適化條件之超臨界CO2萃取，透過分析立體結構、成分以及抗氧化活性，比較酵素輔助超臨界CO2萃取對CPH及其萃取物之影響。結果顯示，以R-F輔助超臨界CO2萃取CPH獲得最高總類黃酮為27.53±1.17 mg/g，其DPPH及ABTS自由基清除能力之IC50分別為53.28±0.03 μg/mL和38.63±0.16 μg/mL，均顯著低於其他處理，且可提高總兒茶素含量，其中EGC含量增加幅度最大，是單獨使用超臨界CO2萃取含量的2.7倍。

After cocoa processing, the remaining cocoa pod husks (CPH) account for about 74% of the total weight. Studies have shown that CPH is rich in phenolic compounds, which are considered the as primary components with antioxidant activity. Supercritical carbon dioxide (SC-CO2) extraction includes many advantages such as low operating temperature, high diffusion capacity, non-polluting, etc. It has become one of the primary choices for extraction of high-value natural products. Phenolic compounds are mostly ester-bound to plant cell walls. To increase the release of phenolic compounds from plant cells, the objective of this study was to establish an enzyme-assisted SC-CO2 extraction technology for CPH. The experiment was divided into two stages. First, CHP was dried with hot air and ground into powder. A four-factor, three-level Box-Behnken reaction surface method was applied to optimize the extraction conditions of CPH powder by SC-CO2. A second-degree polynomial equation was established. The optimal extraction conditions were 61℃, 4735 psi, 17.4% co-solvent ratio extract for 118 minutes. Second, fresh CPH puree was treated with four enzymes individually, including RAPIDASE® FIBER (R-F), RAPIDASE® TF, BREWERS COMPASSTM and VALIDASE® TRL. Different enzyme concentrations (0.5-3.0%) and reaction time (0-6 hr) were used for hydrolysis at 50℃, pH 5.5. The changes of reducing sugars and total phenolic content were determined. Finally, CHP was hydrolyzed by enzymes, and followed by SC-CO2 extraction under optimal conditions. Effects of enzyme-assisted SC-CO2 extraction on CHP were evaluated by the structure, composition and antioxidant activity. The results showed that the highest total flavonoids content obtained by R-F-assisted SC-CO2 extraction of CPH were 27.53±1.17 mg/g. The IC50 of DPPH and ABTS free radical scavenging capabilities were 53.28±0.03 μg/mL and 38.63±0.16 μg/mL, respectively, which were significantly lower than other treatments. That total catechin content also increased. EGC content was 2.7 times higher than using supercritical CO2 extraction alone.

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