檸檬籽之抑菌性研究及天然抑菌劑之開發

本研究以洋菜擴散法分析檸檬籽萃取液對Escherichia coli ATCC 8739、Bacillus subtilis NPUST-UN0N01、Pichia guilliermondii NPUST Y23之抑菌活性及使用肉湯稀釋法分析檸檬籽萃取液對Aspergillus niger NPUST-UN-Asp01之抑菌活性。由研究結果顯示，檸檬籽以 30 ℃ 震盪之95 %乙醇萃取液具有較佳之抑菌性。進一步探討溫度、pH、金屬離子及常用食品化學添加物等對檸檬籽95 %乙醇萃取液抑菌活性之影響，萃取液經121℃ 加熱20分鐘處理後，仍具抑菌活性；檸檬籽萃取液會受pH值改變而影響抑菌活性；檸檬籽95 %乙醇萃取液含有1 % Zn^(2+)離子明顯提高檸檬籽萃取液之抑菌活性；檸檬籽95 %乙醇萃取液含有1 % 幾丁聚醣可顯著增加對B. subtilis、E. coli及P. guilliermondii之抑菌性，其抑菌圈分別從14.46 mm、13.22 mm及9.30 mm增加至57.88 mm、50.39 mm及21.56 mm。檸檬籽95%乙醇萃取液對試驗菌株最小抑菌濃度為0.5~8 %。在儲藏性試驗中，不同儲存溫度下（4 ℃、37 ℃ 及 60 ℃），檸檬籽95%乙醇萃取液仍保有其抑菌活性。不同濃度自製檸檬籽95%乙醇萃取抑菌劑及市售抑菌劑（NK-1），對試驗菌株之抑菌效果，當自製檸檬籽95%乙醇萃取抑菌劑濃度為1 % 時，其對B. subtilis及E. coli之抑菌率與1 %市售抑菌劑（NK-1）相同，達100 % 抑菌率，對P. guilliermondii之抑菌率為89.62 %；當自製檸檬籽95 %乙醇萃取抑菌劑濃度為0.2 %時，對E. coli及P. guilliermondii之抑菌率可達100 %，與0.2 %市售抑菌劑（NK-1）相同，而對B. subtilis之抑菌率為83.71 %，大於0.2 %市售抑菌劑（NK-1）之抑菌率64.02 %。檸檬籽95 %乙醇萃取液對試驗菌株之抑菌模式皆為靜菌作用，無法殺滅細菌。將檸檬籽95 % 乙醇萃取液經膠體過濾層析分離純化後具抑菌活性之區分LSE-F2分析鑑定其成分，結果發現其主要為橙皮苷與柚皮苷。小鼠正常肝細胞FL83B及人類肝癌細胞Hep G2經不同濃度之檸檬籽95 % 乙醇萃取液（2-8 mg/mL）處理24小時後，檸檬籽95 % 乙醇萃取液對小鼠正常肝細胞FL83B細胞均不被檸檬籽95 % 乙醇萃取液影響其生長；而Hep G2細胞隨檸檬籽95 % 乙醇萃取液濃度增加呈現不同程度之皺縮、飄起，其存活率隨檸檬籽萃取液濃度增加而降低，於檸檬籽萃取液濃度為7 mg/mL時，Hep G2之存活率皆低於50 %。顯示出檸檬籽95 % 乙醇萃取液具有保護正常肝細胞，抑制人類肝癌細胞Hep G2生長存活之效果。

關鍵字

洋菜擴散法；檸檬；檸檬籽；抑菌活性

並列摘要

In this study, the antibacterial activity of lemon seed extracts against Escherichia coli ATCC 8739, Bacillus subtilis NPUST-UN0N01 and Pichia guilliermondii NPUST Y23 was analyzed by the method of agar diffusion, and the antibacterial activity of lemon seed extracts against Aspergillus niger NPUST-UN-Asp01 was analyzed by broth dilution method. According to the results of the study, the lemon seed extracts has the best antibacterial properties at 30 °C with 95 % ethanol using the shaking extraction method. The study investigated the effects of temperature, pH value, metal ions, and common food chemical additives on the antibacterial activity of lemon seed 95 % ethanol extract. The extract was treated up to 121 °C for 20 minutes and still maintained antibacterial activity, and was affected by the pH value. The lemon seed 95 % ethanol extract contains 1 % Zn^(2+) ions has the most significant effect of antibacterial activity. The lemon seed 95 % ethanol extract contains 1% chitosan has the most significant effect of antibacterial activity on B. subtilis, E. coli and P. guilliermondii. The diameters of growth inhibition increase from 14.46 mm, 13.22 mm and 9.30 mm to 57.88 mm, 50.39 mm and 21.56 mm, respectively. The minimum inhibitory concentration of lemon seed 95 % ethanol extract against the test strain was 0.5-8 %. In storage tests, lemon seed 95 % ethanol extract still retained its antibacterial activity at different storage temperatures (4 °C, 37 °C, and 60 °C). When the concentration of lemon seed 95% ethanol extract preservative is 1 %, the inhibition ratio was up to 100% on B. subtilis and E. coli. The inhibition ratio was 89.62 % on P. guilliermondii; When the concentration of lemon seed 95 % ethanol extract preservative was 0.2 %, the inhibition ratio was 100 % on E. coli and P. guilliermondii. The inhibition ratio was 89.62 % on P. guilliermondii. The antimicrobial was better than commercial preservative. The effect of lemon seed 95 % ethanol extract on test microbial strains was bacteriostatic not germicidal. The lemon seed 95% ethanol extract substance are mainly hesperidin and naringin and it identified by LC-MS/MS. After treatment of FL83B and Hep G2 with different concentrations of lemon seed 95 % ethanol extract (2-8 mg/mL) for 24 hours, lemon seed 95 % ethanol extract didn’t affect the growth of FL83B cells; Hep G2 cells showed different degrees of shrinkage and floating. The cell viability decreased with the increase of the concentration of lemon seeds extracts. When the concentration of lemon seeds extracts was 0.7 %, the cell viability of Hep G2 was less than 50 %. It showed that the lemon seed 95% ethanol extract has the effect of protecting normal liver cells and inhibiting the growth and survival of Hep G2 cells.