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研究生:陳盈潔
研究生(外文):Ying Jie Chen
論文名稱:梔子萃取液之抑菌及抗氧化性之探討
論文名稱(外文):Antibacterial and Antioxidatives Activities of Gardenia Fruit Extract
指導教授:謝寶全謝寶全引用關係
指導教授(外文):Pao-Chuan Hsieh
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:223
中文關鍵詞:洋菜擴散法梔子抑菌活性抗氧化活性
外文關鍵詞:agar diffusion methodGardenia fruitantibacterial activityantioxidation
相關次數:
  • 被引用被引用:32
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中文摘要
學號:M8936015
論文名稱:梔子萃取液之抑菌及抗氧化性之探討 總頁數:223
學校名稱:國立屏東科技大學 系(所)別:食品科學系(所)碩士班
畢業時間及摘要別:九十年度第二學期碩士學位論文摘要
研究生:陳盈潔 指導教授:謝寶全 博士
論文摘要內容:
本研究系探討藥用植物梔子果實之抗菌及抗氧化活性,將梔子以蒸餾水萃取,並添加果膠分解酵素製備,即取得含抑菌成分之萃取液,利用洋菜擴散法分析梔子萃取液之抑菌活性,結果以梔子水萃取液添加果膠分解酵素對實驗中9株試驗菌株具有廣泛的抑菌活性,進一步探討梔子萃取液在各種pH、溫度、金屬離子及常用化學添加物,對其抑菌活性之影響,結果發現,梔子萃取液不受pH值改變而降低其抑菌活性;對加熱處理及在酸性條件下,其抑菌活性不受影響,添加金屬離子Zn2+、Ca2+、Fe3+、Mg2+會使梔子萃取液對Bacillus subtilis及Flavobacterium sp.之抑菌活性降低,梔子萃取物對革蘭氏陽性菌(例如:李斯特菌)之抑菌活性大於革蘭氏陰性菌(例如:大腸桿菌)。於液體培養基中,添加2 ﹪之梔子萃取液對金黃色葡萄球菌及大腸桿菌之抑制率分別為:100及97.8 ﹪。添加0.1 ﹪幾丁聚糖及脂肪酸蔗糖酯於梔子萃取液中,並不會對梔子萃取液抑菌活性有影響;梔子中所含之生理活性物質geniposide並不具抗菌效果,而梔子色素部分需大於10 ﹪含量始具有抗菌效果;而不論梔子中之生理活性物質或是色素部分,對清除超氧陰離子及DPPH自由基皆具有效果,清除率分別為40.5、53.5 ﹪及29.2、58.5 ﹪,此二物質抗氧活性為43.2 ﹪及76.4 ﹪。添加0.5 ﹪之梔子萃取液,於4℃條件下貯存的奶茶中,貯存4天,其生菌數皆低於控制組。
以膠體濾層析進行初步純化後,其抑菌活性皆高於原萃取液,初步測其抗菌成分之分子量為325.7 Da,以掃描式電子顯微鏡探討梔子水萃取液之初步抑菌模式,結果顯示添加梔子萃取液後菌體之細胞壁皺縮及破裂,此可能為梔子水萃取液抑菌之原理。經過氣相層析質譜儀測定梔子水萃取液中所含之抑菌物質可能為hexadecamide 及 cis-9-octadecenamide.
關鍵詞:洋菜擴散法、梔子、抑菌活性、抗氧化活性
ABSTRACT
The purpose of this research was to evaluate the antibacterial and antioxidative activities of Gardenia fruit. The active ingredient was prepared by extracting the Gardenia fruit with distilled water with adding pectinase. The antibacterial tests were carried out by using agar diffusion method. Results revealed that the extract of Gardenia fruit had an extensive antibacterial activities on 9 microbial strains. The effect of pH, temperature, metal ions and chemical additives on antibacterial activity of Gardenia fruit was tested. The antibacterial activity did not decreased with an increase of pH and temperatures. Adding metal ions especially Zn2+, Ca2+, Fe3+ and Mg2+ in Gardenia fruit extract showed that antibacterial activity against Bacillus subtilis and Flavobacterium sp. decreased. The Gardenia fruit extract inhibited the growth of Gram-positive bacteria (such as Listeria monocytogenes) more than Gram-negative bacteria (such as Escherichia coli). Addition of 2 % Gardenia fruit extract into the broth of Staphylococcus aureus and Escherichia coli, the inhibition ratios were 100 % and 97.8 %, respectively. Adding the chitosan lactate and fatty acid ester in Gardenia fruit extract showed no effect on the antibacterial activity of Gardenia fruit extract. The ingredient that had physiological activity of Gardenia fruit was geniposide, with no antibacterial activity. Althought the antibacterial activity of geniposide and pigment less than 10 % from Gardenia fruit were low; however, they still had antioxidation activity. Geniposide and pigment of Gardenia fruit scavenged of superoxide by 40.5 and 53.5 %, and scavenged DPPH radicals by 29.2 and 58.5 %, respectively. The geniposide and pigment could inhibit the peroxidation at the level of 43.2 and 76.4 %, respectively. For food application, adding of 0.5 % Gardenia fruit extract to milk-tea showed less microbial counts than that of control after five days storage. By using gel filtration (Sephadex G-25, Sephadex G-15), The partial purified fraction showed higher antibacterial activity than crude extract. The molecular weight of this fraction was identified to be 325.7 Da. Using scanning electron microscopy to check the mode of antibacterial activity of Gardenia fruit water extract. The result showed the cell wall of bacteria were wrinkled and broken after added Gardenia fruit water extract, maybe it was the principle of antibacterial activity of Gardenia fruit water extract. And then determined the compounds of antibacterial activity in Gardenia fruit water extract by GC-mass. The result showed the compounds maybe were hexadecamide and cis-9-octadecenamide.
Key words: agar diffusion method, Gardenia fruit, antibacterial activity, antioxidation activity.
目錄
===========================
中文摘要 I
英文摘要 III
謝誌 IV
目錄 VI
表目 XIV
圖目 XVⅡ
壹、前言 1
貳、文獻回顧 4
一、抗菌物質之探討 4
(一)食品保存劑之特性 4
(二)化學合成之食品保存劑 6
(三)天然抗菌物質 7
(四)食品中自然存在的抗菌物質 30
(五)存在中藥之抗菌物質 45
(六)選用試驗材料之特性 51
二、抗氧化物質之探討 57
(一)脂質氧化反應 58
(二)抗氧化劑的作用機制 61
(三)天然抗氧化劑 63
參、材料與方法 70
一、試驗材料 70
二、實驗方法 75
1. 梔子抗菌物質的萃取與製備 75
2. 抗菌性物質的製備 77
3. 梔子標準品geniposide之抽取、分離及精製 78
4. 梔子中戊乙醯去羥梔子苷
(penta acetyl geniposide)之製備 78
5. 梔子色素之抽取 80
6. 比較添加市售果膠酵素去除果膠對梔子萃取液
之抗菌影響 81
7. 梔子成分分析 81
(1)果膠定量法 81
(2)還原醣量之測定 81
(3)梔子萃取液之糖類分析 83
(4)天然色素色層層析分析 83
(5)胡蘿蔔素之測定 84
(6)梔子萃取液中總酚類含量之測定 85
(7)梔子萃取液中總類黃酮物質含量之測定
(AlCl3 呈色法) 86
8. 菌種之保存與更新 86
9. 試驗菌株之製備 87
10. 抑菌試驗 87
11. 抑菌率測定 88
12. 黴菌菌絲重之測定 88
13. 生菌數測定 89
14. 萃取時間對梔子萃取液之抑菌活性影響 89
15.pH值對梔子萃取液抑菌活性影響 89
16.不同稀釋濃度之梔子萃取液對其抑菌活性
之影響 90
17.金屬離子對梔子萃取液之抑菌活性影響 90
18.食品添加劑對梔子萃取液之抑菌活性影響 90
(1) 化學試劑 90
(2) 安定劑 91
(3) 抗氧化劑 91
19. 梔子萃取液與抗生素抑菌活性之比較 91
20. 不同濃度之梔子萃取液及己二烯酸鉀
(potassium sorbate)對Staphylococcus aureus
生長之影響 92
21. 貯存條件對梔子萃取液抑菌活性之影響 92
22. 活性碳吸附處理對梔子萃取液抑菌活性之影響 92
23. 添加幾丁聚醣乳酸鹽類對梔子萃取液抑菌性
之影響 92
24. 添加脂肪酸蔗糖酯對梔子萃取液抑菌性之影響 93
25. 與日本商品化食品保存劑抑菌性之比較 93
26. 低溫沈澱試驗 93
27. 甲醇區分試驗 94
28. 最低抑菌濃度
(minimum inhibitory concentration,
MIC)測定 94
29. 梔子萃取液添加於市售奶茶之抑菌貯存
安定性試驗 94
30. 梔子萃取液與商品化之梔子色素之
抑菌活性比較 95
31. 抑菌模式之初步探討 95
(1) 添加半胱胺酸於梔子萃取液之抑菌影響 95
(2) SEM之檢測 95
32. 梔子之抗氧化能力試驗 96
(1) 抗氧化試驗 96
(2) 抗氧化試驗之捕捉
1,1-diphenyl-α,-picrylhydrazyl
(DPPH) 自由基能力測定 97
(3) 捕捉超氧陰離子能力測定 98
33. Amberlite XAD-7管柱層析 98
(1)Amberlite XAD-7樹脂前處理 98
(2)Amberlite XAD-7樹脂分析 99
34. 膠體過濾色層分析
(gel filtration chromatography)
進行萃取液之初步分離純化 99
35. 以Sephadex G-15膠體過濾法測定梔子萃取液
中抑菌物質之分子量 100
36. 梔子抑菌成分分離純化流程 100
37. 統計分析
(statistical analysis system,SAS) 101
肆、結果討論 103
一、梔子萃取液對試驗菌株抑菌活性之探討 103
(一) 不同溶劑萃取液對試驗菌株抑菌活性
之影響 103
(二)與傳統熬煮中藥法之抑菌性比較 106
(三)梔子依序以三種不同極性溶劑進行連續
萃取,檢測其抑菌物質之極性分佈 109
(四) 不同溶劑迴流萃取梔子粉末之抑菌
影響 112
(五)梔子以不同pH值醋酸緩衝溶液萃取之
抑菌影響 114
(六) 乙醇區分試驗 116
(七) 低溫沈澱試驗 116
(八) 梔子生理活性物質(geniposide)及
戊乙醯去羥梔子苷(penta-acetyl
geniposide) 抗菌性之探討 119
(九) 梔子色素抗菌性之探討 122
(十) 添加果膠分解酵素對梔子萃取液之
抗菌活性影響 124
(十一)果膠分解酵素於不同pH下之抗菌性
影響 126
(十二)不同濃度之果膠分解酵素對
梔子萃取液之抑菌活性之影響 128
二、萃取液之特性探討 130
(一)梔子萃取液經高溫高壓處理對其抑菌活
性之影響 130
(二)梔子萃取液經不同pH處理對其抑菌
活性之影響 132
(三)金屬離子對梔子萃取液抑菌活性之影響 134
(四)食品添加劑對梔子萃取液抑菌活性之
影響 137
1.化學試劑 137
2.安定劑 140
3.抗氧化劑 141
(一) 活性碳吸附對梔子萃取液抑菌活性之
影響 146
(六)不同濃度的梔子萃取液對試驗菌株之
抑菌率 146
(七)梔子萃取液對黴菌抑制率之測定 151
(八)不同濃度之梔子萃取液及己二烯酸鉀對
S. aureus生長之影響 153
(九)貯存條件對梔子萃取液抑菌活性之影響153
(十)不同稀釋度之梔子萃取液對其抑菌活性
之影響 156
(十一)梔子萃取液與抗生素之抑菌活性比較156
三、梔子水萃取液之成分分析 159
(一)梔子水萃取液中果膠定量 159
(二)梔子水萃取液中還原醣含量之測定 159
(三)梔子萃取液之醣類分析 160
(四)色素中胡蘿蔔素之測定 162
(五)梔子萃取液中總酚類化合物及總類黃酮
之測定 163
四、梔子萃取液抑菌作用之探討 163
(一) 梔子萃取液對試驗菌株之靜菌或殺菌
作用 163
(二)梔子萃取液之最小抑菌濃度 164
五、探討添加具抑菌活性物質於梔子萃取液中,
對其抑菌活性之影響 167
(一) 添加幾丁聚醣乳酸鹽
(chitosan lactate)對梔子萃取液
抑菌活性之影響 167
(二) 添加脂肪酸蔗糖酯(sugar ester)
對梔子萃取液之抑菌活性影響 169
六、與商品化之梔子色素抑菌活性比較 171
七、與商品化天然食品保存劑抑菌性之比較 173
八、梔子萃取液應用於食品中之抑菌活性探討 173
九、梔子色素及geniposide之抗氧化分析 175
(一)梔子之geniposide及色素對
1,1-diphenyl-α-β-picrtylhydrazyl
(DPPH)自由基之捕捉效應 177
(二)梔子之geniposide及色素對超氧陰離子
(Superoxide anion, O2-)之捕捉效應 177
(三)梔子之geniposide及色素之抗氧化
能力試驗 181
十、梔子萃取液之抑菌物質分析 181
(一)梔子萃取液之層析分離及其抑菌活性之
影響 183
(1)Amberlite XAD-7 管柱層析 183
(2)梔子萃取液Sephadex G-25膠體層析 183
(3)梔子萃取液之 Sephadex G-15膠體
層析 187
(4)GC-mass 分析 187
十一、梔子萃取液中抑菌物質分子量之初步分析 187
十二、梔子萃取液抑菌機制之初步分析 190
(一) 添加半胱胺酸對梔子抑菌性之
探討 190
(二) 以SEM探討梔子萃取液對菌體
之作用 193
伍、結 論 196
陸、參考文獻 200
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