臺灣博碩士論文加值系統

本研究以中國大陸內蒙古地區產之甘草為材料，探討極性不同溶劑萃取物之抑菌圖譜，甲醇萃取物之抑菌活性，並對甲醇萃取物之抑菌成分分離純化，純化之抑菌化合物進行抑菌性質測定。
中國大陸內蒙古地區產之甘草分別以水、甲醇、乙酸乙酯及二氯甲烷為萃取溶劑，分析萃取物之抑菌圖譜。結果顯示甲醇萃取物具有最佳之抑菌圖譜，在16株測試細菌中，抑制10株G(+)及2株G(-)，其中對B. subtilis CCRC 10267及Staph. aureus CCRC 12657具有最低抑制濃度 (0.05﹪)。添加0.07﹪甘草甲醇萃取物至接種Staph. aureus CCRC 12657之TSB培養基中，結果顯示0.07﹪甘草甲醇萃取物即具殺菌作用，培養液之菌落數在2 h內由106 CFU/mL急速降至0 CFU/mL，並維持0 CFU/mL至48 h。甘草甲醇萃取物之殺菌作用在pH 5~10的環境中相當穩定，於添加培養8 h後，各培養液之菌落數均維持0 CFU/mL。甘草甲醇萃取物的殺菌作用對熱相當穩定，添加0.07﹪經121℃熱處理15分鐘之萃取物至接種Staph. aureus CCRC 12657之TSB培養基中，培養液的菌落數在36 h內均維持0 CFU/mL。於Staph. aureus CCRC 12657生長靜止期 (109 CFU/mL) 加入0.04~0.10﹪甘草甲醇萃取物，結果顯示在添加後之37 h內，培養液之菌落數均與對照組相似：109 CFU/mL，不具有殺菌或抑菌作用。
接著進行甘草甲醇萃取物抑菌成分之分離純化。首先將甘草甲醇萃取物以Amberlite XAD-7管柱層析，依序以水-甲醇(v/v) = 6:4(Ⅰ)、3:7(Ⅱ)、2:8(Ⅲ)、1:9(Ⅳ)、0:10(Ⅴ)及甲醇-乙酸乙酯(v/v) = 9:1(Ⅵ)、8:2(Ⅶ)等七個比例為流析溶液，以UV254nm進行光譜偵測及抑菌活性分析。結果顯示區分Ⅲ~Ⅴ具有抑菌活性，活性之回收率分別為29.5﹪、52.5﹪及3.7﹪。接著將區分Ⅲ以Sephadex LH-20進行膠質穿透層析，以水-甲醇 = 0:100→18:82沖提液梯度沖提，以UV254nm光譜偵測，並分析抑菌活性。結果得到區分Ⅲ-A~F六個具有抑菌活性之區分，其中以區分Ⅲ-D表現出最高之抑菌活性，回收率為2.7﹪；HPLC之圖譜顯示，區分Ⅲ-D僅在RT 27.60min及41.68min呈現二個主要的吸收峰，含有二個主要化合物。區分Ⅳ同樣地以Sephadex LH-20進行膠質穿透層析，以水-醋酸-甲醇 = 29:1:70→3:1:96之流析液梯度沖提，經UV254nm光譜偵測及活性分析之結果得到活性區分Ⅳ-A，抑菌活性之回收率為6.0﹪，HPLC之圖譜顯示，僅在RT 42.16min呈現主要之吸收峰，以積分面積計約佔70﹪。
在11株G(+)及5株G(-)之食品污染菌中，區分Ⅲ-D與區分Ⅳ-A均抑制10株G(+)及1株G(-)，但抑菌環直徑平均以區分Ⅲ-D較區分Ⅳ-A大。區分Ⅲ-D對Staph. aureus CCRC 12657具有最低之抑制濃度：5μg/mL；區分Ⅳ-A對B. cereus CCRC 10250、B. subtilis CCRC 10267、Staph. aureus CCRC 12657及Staph. aureus CCRC 12653具有最低之抑制濃度：20μg/mL。

In this study, antimicrobial spectrum of various solvent extracts, antimicrobial properties of methanol extracts, isolation and purification of antimicrobial components of methanol extracts, structure identification of antimicrobial components and antimicrobial properties of purified or partial purified antimicrobial components from Glycyrrhiza radix cultivated on Mongolia of China were studied.
Among the four extracts of licorice, methanol extracts showed the best antimicrobial spectrum which inhibited the growth of ten strains of G(+) and two strains of G(-) among the sixteen tested bacteria. Bacillus subtilis CCRC 10267 and Staph. aureus CCRC 12657 showed the lowest minimun inhibitory concentration, 0.05%. Methanol extracts showed bactericidal action. When 0.07% methanol extracts added to TSB medium which inoculated Staph. aureus CCRC 12657, the colony of Staph. aureus CCRC 12657 was decreased rapidly from 106 CFU/mL to 0 CFU/mL in 2h and maintained at least 48h. At the stationary phase of Staph. aureus CCRC 12657, 0.04%~0.10% methanol extracts were added to the broth, the colonies of the strain were the same with the control (109 CFU/mL) during 37h after addition. It was neither bactericidal nor bacteriostatic action. The bactericidal action of methanol extracts of licorice was very stable for pH treatment. Colonies of Staph. aureus CCRC 12657 were maintained 0 CFU/mL at pH 5~10 after 8h cultivation. The bactericidal action of methanol extracts of licorice was very stable for heat treatments. Methanol extracts of licorice were treated at 121℃ for 15min and added (0.07%) to the TSB medium, the colonies of Staph. aureus CCRC 12657 maintained 0 CFU/mL within 36h.
And then, isolation and purification of antimicrobial components of methanol extracts from licorice were studied. At first, methanol extracts of licorice was subjected to Amberlite XAD-7 column chromatography, eluted with water-methanol (v/v) = 6:4 (Ⅰ), 3:7 (Ⅱ), 2:8 (Ⅲ), 1:9 (Ⅳ), 0:10 (Ⅴ) and methanol-ethyl acetate (v/v) = 9:1(Ⅵ), 8:2 (Ⅶ) and monitored at OD254. Among seven spectral fractions, fraction Ⅲ, Ⅳ and Ⅴ showed antimicrobial activity. The recoveries of activity were 29.5﹪、52.5﹪ and 3.7﹪, respectively. Fraction Ⅲ was followed by Sephadex LH-20 gel permeation chromatography, eluted with gradient of water-methanol = 0:100 to 18:82 and monitored at OD254. Seven spectral fractions were fractionationated, fraction Ⅲ-A to F showed antimicrobial activity. The recoveries of activity were 1.35%, 0.68%, 1.01%, 2.7%, 1.35% and 0.34%. There were two retention times of fraction Ⅲ-D on HPLC spectrum that indicated two major compounds to be present in the fraction Ⅲ-D. Fraction Ⅳ was also followed by Sephadex LH-20 gel permeation chromatography, eluted with gradient of water-acetic acid-methanol = 29:1:70→3:1:96 and monitored at OD254. Fraction Ⅳ-A showed antibacterial activity and the recovery of activity was 6.0﹪. There was only one retention time of fraction Ⅳ-A on HPLC spectrum. It was indicated a major compound to be present in fraction Ⅳ-A and the percentage of the peak was about 70﹪by the area-calculation.
Both fraction Ⅲ-D and fraction Ⅳ-A inhibited the growth of ten strains of G(+) and one strain of G(-) among the sixteen tested bacteria. The average diameter of inhibitory zones of fraction Ⅲ-D were larger than fraction Ⅳ-A. Staph. aureus CCRC 12657 showed the lowest minimun inhibitory concentration of fraction Ⅲ-D, 5μg/mL. B. cereus CCRC 10250, B. subtilis CCRC 10267, Staph. aureus CCRC 12657 and Staph. aureus CCRC 12653 showed the lowest minimun inhibitory concentration of fraction Ⅳ-A, 20μg/mL.

表次…………………………………………………………………… Ⅴ
圖次…………………………………………………………………… Ⅵ
中文摘要……………………………………………………………… 1
英文摘要……………………………………………………………… 3
壹、 前言…………………………………………………………… 6
貳、 文獻整理……………………………………………………… 7
一、 食品保存劑簡介……………………………………………… 7
(一) 食品保存劑特性………………………………………….. 7
(二) 食品保存劑來源………………………………………….. 9
(三) 食品保存劑之抑菌機制………………………………….. 18
二、 甘草簡介……………………………………………………… 22
(一) 名稱……………………………………………………….. 22
(二) 基原、種類及分布……………………………………….. 22
(三) 植物性狀………………………………………………….. 24
(四) 藥材特徵………………………………………………….. 24
三、 甘草之組成分………………………………………………… 24
四、 甘草之藥理作用……………………………………………… 26
五、 甘草之生理活性……………………………………………… 29
六、 甘草之毒理評估……………………………………………… 34
七、 甘草抑菌作用之相關研究…………………………………… 34
八、 甘草之抑菌機制……………………………………………… 36
參、 材料與方法…………………………………………………… 39
一、 材料…………………………………………………………… 39
二、 方法…………………………………………………………… 40
第一部份：甘草萃取物抑菌活性之探討……………………… 40
(一) 甘草抑菌萃取物之製備……………………………… 40
(二) 抑菌活性試驗測定法……………………………… 41
1. 洋菜擴散法………………………………………… 41
2. 洋菜稀釋法…………………………………………… 42
(三) 抑菌性質測定………………………………………… 42
1. 甘草甲醇萃取物對Staph. aureus CCRC 12657
生長之影響…………………………………………… 42
2. 甘草甲醇萃取物對Staph. aureus CCRC 12657
生長靜止期之影響…………………………………… 43
3. 甘草甲醇萃取物抑菌活性對pH之穩定性…………. 43
4. 甘草甲醇萃取物抑菌活性對熱之穩定性…………… 44
第二部分：甘草甲醇萃取物抑菌成分分離純化之研究………. 44
(一) 甘草甲醇萃取物之製備……………………………… 44
(二) 甘草甲醇萃取物對分離膠體之吸附試驗…………… 44
(三) 抑菌活性分析~臨界稀釋法…………………………. 45
(四) Amberlite XAD-7管柱層析…………………………. 46
(五) Sephadex LH-20膠質穿透層析……………………... 47
(六) 高效能液相層析……………………………………… 48
第三部分：甘草抑菌區分抑菌性質之探討…………………… 49
(一) 粉末樣品之製備…………………………………….. 49
(二) 抑菌活性試驗………………………………………… 49
1. 洋菜擴散法…………………………………………… 49
2. 洋菜稀釋法…………………………………………... 50
肆、 結果與討論……………………………………………………. 51
第一部份：甘草萃取物抑菌活性之探討…………………………… 51
(一) 不同溶劑甘草萃取物之抑菌圖譜…………………………… 51
(二) 甘草甲醇萃取物之抑菌性質………………………………… 51
1. 對各測試菌株之抑菌環直徑及最低抑制濃度…………… 51
2. 甘草甲醇萃取物對Staph. aureus CCRC 12657
生長之影響………………………………………………… 54
3. 甘草甲醇萃取物對Staph. aureus CCRC 12657
生長靜止期之影響………………………………………… 54
4. 甘草甲醇萃取物抑菌活性對pH之穩定性……………… 56
5. 甘草甲醇萃取物抑菌活性對熱之穩定性………………… 56
第二部分：甘草甲醇萃取物抑菌成分分離純化之研究…………… 59
(一) 膠體選擇……………………………………………………… 59
(二) Amberlite XAD-7管柱層析………………………………… 59
(三) Sephadex LH-20膠質穿透層析……………………………… 63
第三部份：甘草抑菌區分抑菌性質之探討………………………… 68
(一) 甘草抑菌區分之抑菌圖譜…………………………………… 68
(二) 甘草抑菌區分對各測試菌株之抑菌環直徑
及最低抑制濃度……………………………………………… 68
伍、 結論……………………………………………………………… 74
陸、 參考文獻………………………76

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