- 學位論文
超微細化研磨技術對竹葉萃出物類黃酮含量 及抗菌活性之影響
Effect of ultrafine milling on total flavonoids content and antimicrobial activity of bamboo leaf extracts
摘要
中文摘要 本研究利用超微細化研磨技術進行竹葉之微細化及萃取，探討研磨後的竹葉濾渣之粒子粒徑、粗萃物之化學成分與生物活性物質，以及竹葉粗萃液對細菌生長抑制能力之評估，並針對竹葉中抑菌物質進行初步判定。試驗中所使用之研磨萃取溶劑包括蒸餾水及60%乙醇。結果顯示，在水或60%乙醇中研磨竹葉1 hr後，其平均粒徑及d90顯著下降，延長研磨時間，其粒徑雖持續降低但趨勢漸緩，4 hr研磨後，竹葉樣品平均粒徑為12 μm，d90為25 μm，已達細胞破壁之目標。以水為溶劑，經研磨2 hr之竹葉，其水粗萃液之可溶性糖及總類黃酮含量顯著提高，但總酚類含量並無顯著差異。當以60%乙醇為溶劑進行研磨萃取時，可溶性糖含量減少，但總類黃酮含量顯著提高為1.76 mg catechin equivalent /g竹葉粗粉，顯示60%乙醇為總類黃酮之良好萃取溶劑。乙醇粗萃液之DPPH自由基清除力高於水粗萃液。每克竹葉粗粉含有總氰氫酸含量相當於2.5 μg HCN，經超微細化處理後，竹葉濾渣顆粒之總氰氫酸含量提高為3-4 μg HCN；而竹葉粗萃液之總氰氫酸含量則降低為0.17-0.21 μg HCN，且不受超微細化處理之影響。利用矽膠管柱進行粗萃液初步分離，以高效液相層析進行70%甲醇沖提液之組成分析，結果顯示有三個主要吸收峰，依200-400 nm之UV掃描吸收光譜圖，可能為類黃酮化合物，經質譜儀判定其中兩個吸收峰所代表之化合物可能為異葒草苷或葒草苷及異牡荊苷或牡荊苷。竹葉粗萃物進行抑菌試驗之結果顯示，於濾紙擴散法、孔盤擴散法及序列稀釋法試驗中，以60%乙醇研磨萃取之竹葉粗萃液10倍稀釋倍率對Bacillus cereus及Staphylococcus aureus的生長有顯著的抑制效果，但對Escherichia coli及Salmonella typhimuriun菌之生長卻無顯著抑制效果。在12 hr內，10倍稀釋倍率之乙醇粗萃液便可抑制菌落數增加。以菌落之大小能比較出竹葉乙醇萃液對S. aureus之抑制濃度。而以水為研磨萃取溶劑之竹葉粗萃物，即使以原粗萃液之含量，對本實驗4種測試菌種則均無抑制生長之效果。整體而言，臺灣具有豐富的竹類資源，而竹葉以60%乙醇作為萃取溶劑及超微細化研磨處理有助於提升其類黃酮含量及其對Bacillus cereus 及 Staphylococcus aureus的抑菌活性，因此，竹葉粗萃液作為有抑菌作用之食品添加物之潛力。
並列摘要
Abstract This study applied ultrafine-milling technology on bamboo leaf. The post-milled particle size of bamboo leaf was examined. The chemical content, bioactive material of bamboo leaf extract, and its ability to suppress bacterial growth were investigated. The antimicrobial material in bamboo leaf was also preliminarily determined. The extracting solvent used in this study included water and 60% ethanol. After a 1 hour ultrafine-milling treatment, using either water or 60% ethanol, the mean particle size and d90 of bamboo leaf decreased obviously. With even longer ultrafine-milling treatment, the mean particle size continued to decrease but changed more slowly. After a 4 hour ultrafine-milling treatment, the mean particle size and d90 were 12 μm and 25 μm, reaching the goal of destructed cell wall. After a 2 hour ultrafine-milling treatment with water, the total soluble carbohydrates and total flavonoids of bamboo leaf extract elevated obviously, but the total phenolics showed no apparent differences. Under the same milling condition except using 60% ethanol, the total soluble carbohydrates of bamboo leaf extract decreased, but the total flavonoids increased to 1.76 mg catechin equivalent /g ground bamboo leaf powder(GP), suggesting that 60% ethanol is a good extracting solvent of flavonoids. The DPPH scavenging capacity of ethanol extract was the highest than that of water extract under all the conditions in this study. The result of total cyanide content analysis showed that dried ground bamboo leaf powder contained 2.5 μg HCN. After ultrafine-milling treatment, the total cyanide content of residue of bamboo leaf increased to 3-4 μg HCN/g GP. But the total cyanide content of bamboo leaf extract decreased obviously after rotary evaporation and cetrifuge treatment. Bamboo leaf extract composition analysis performed by silica gel column, 70% methanol elution and High-performance liquid chromatography (HPLC) demonstrated that there were three major peaks on the HPLC chromatograms of bamboo leaf extract. According to the UV scanning spectra of bamboo leaf extract from 200 to 400 nm wavelength, the components were very likely to be flavonoids, while the Mass spectrometry (MS) was determined as isoorientin (or orientin) and isovitexin (or vitexin). Antimicrobial assay was performed by conducting agar diffusion assay, serial dilution method, and hole-plate diffusion method. The results showed that bamboo leaf ethanol extract can apparently suppress the growth of Bacillus cereus and Staphylococcus aureus at the dilution of 10, but it was ineffective for Escherichia coli and Salmonella typhimuriun. Time-kill method showed that bamboo leaf ethanol extract at the dilution of 10 could persist antimicrobial effects within 12 hours. The inhibitory concentration of bamboo leaf ethanol extract for S. aureus was compared by using colony size method. On the contrary, bamboo leaf water extract, even at the original concentration, had no antimicrobial effect for all the 4 kinds of bacteria in this study. Bamboo leaf treated with ultrafine-milling using 60% ethanol as extracting solvent could achieve higher flavonoids content and antimicrobial activity for Bacillus cereus and Staphylococcus aureus. According to this study and considering the fact that there are abundant bamboo resources in Taiwan, bamboo leaf extract has great potential to be excellent food additives as antimicrobial agent.