摘要
以蒸氣蒸餾法萃取柳橙果皮之精油，利用GC分析其成份，主要成份為limonene (859.98 mg/g)。經柳橙果皮中自體酵素作用後能明顯將碳氫?烯類化合物轉換成含氧?烯類化合物。以30℃作用7天，含氧?烯類化合物linalool可達11.24 mg/g。另外添加20 mM Mg2+時，linalool含量由原來11.24增至12.18 mg/g。選擇新鮮且未加Mg2+之柳橙皮精油、經30℃反應7天與添加20 mM Mg2+經30℃反應7天後所萃取之精油對Malassezia furfur做抗菌試驗，發現在相同濃度下，以添加Mg2+所得之精油對Malassezia furfur抑制效果最好。
利用醋酐乙醯化法所製備乙醯化程度為43.9%之水溶性幾丁聚醣對Malassezia furfur做抗菌試驗，1000 ppm的水溶性幾丁聚醣可使菌數比控制組下降約5 Log CFU/mL。添加1000 ppm之水溶性幾丁聚醣於1.0%經Mg2+催化後之精油乳化物中，可使菌數小於1 Log CFU/mL，其抑菌效果比單獨使用精油或幾丁聚醣為佳。

Abstract
Orange peel oil was extracted by steam distillation and the constituents were analyzed by GC. The major component was limonene (859.98 mg/g). In the oil, there was a significant conversion from hydrocarbon terpenes to oxygenated terpenes after reacting with endogenous enzymes. Through endogenous enzymatic reaction at 30℃ for 7 days, the content of linalool which is an oxygenated terpene reached 11.24 mg/g. Further adding 20 mM of Mg2+, the content of linalool increased form 11.24 up to 12.18 mg/g. Fresh orange peel oil with endogenous enzymatic reaction at 30℃ for 7 days was chosen to test its antifungal activity for Malassezia furfur. It was forword that addition of 20 mM of Mg2+ in the oil for the endogenous enzymatic reaction had the best antifungal activity.
Water soluble chitosan, prepared by acetylation with acetic anhydride, had the degree of acetylation of 43.9%. The chitosan was used to do the antifungal test of Malassezia furfur. When the concentration of the chitosan was 1000 ppm, the cell count was about 5 Log CFU/mL lower than the control. Adding 1000 ppm chitosan into 1.0% emulsified oil in which
20 mM of Mg2+ was added increased its antifungal activity for Malassezia furfur. The cell count was less than 1.0 Log CFU/mL. The antifungal activity was better than just either using orange peel oil or chitosan alone.

目 錄
摘要 I
目錄…………………………………………………………………….....III
表目錄……………………………………………………………...……VII
圖目錄………………………………………………………...………...VIII
一、前言 1
二、文獻整理 3
(一)柳橙之簡介 3
(二)精油及其萃取方式 3
1.水蒸氣蒸餾法 3
2.壓榨法 4
3.溶劑萃取法 4
4.超臨界二氧化碳萃取法 4
(三)植物體中類?烯類化合物的生合成途徑 5
(四)柳橙精油之組成成份 6
(五)精油組成成份中limonene的轉換 6
(六)精油的應用 8
(七)頭皮屑與皮屑芽胞菌 9
(八)目前市售洗髮精之抗屑作用機制 12
(九)添加茶樹精油於洗髮精中的芳香療法 13
(十)精油抗皮屑芽胞菌(Malassezia furfur)之效果 13
(十一)水溶性幾丁聚醣之製備 14
(十二)幾丁聚醣之應用 15
(十三)幾丁聚醣抗菌機制及效果 15
(十四)幾丁聚醣與精油抗菌效果之加乘性 16
三、實驗方法與材料 17
(一) 實驗材料 17
1.樣品 17
2.標準品 17
3.藥品 17
4.菌株 18
5.培養基組成成份 18
6.層析管柱 19
7.透析膜 19
8.儀器裝置 19
(二) 實驗方法………………...……………………………………21
1.柳橙皮精油之萃取 21
2.柳橙皮精油成份分析 21
3.柳橙皮經自體酵素反應後精油成份之改變 23
4.水溶性幾丁聚醣之製備 24
5.水溶性幾丁聚醣乙醯化程度之測定 24
6.抗菌活性測試 28
7.統計分析 31
四、結果與討論 32
(一) 柳橙皮精油之組成 32
(二) 柳橙皮經自體酵素反應後精油成份之改變 33
1. 溫度與時間之影響 33
2. 添加金屬離子溶液之影響 35
(三) 柳橙皮精油乳化物對Malassezia furfur之抗菌活性影響 36
1. 皮屑芽胞菌(Malassezia furfur)生長曲線 36
2. 柳橙精油乳化物抗菌試驗 37
(四) 水溶性幾丁聚醣對Malassezia furfur之抗菌活性 38
1. 水溶性幾丁聚醣濃度之影響 38
2. 添加幾丁聚醣於柳橙皮精油之抗菌活性 39
五、結論 41
六、參考文獻 42

表目錄
表一、柳橙皮精油主要成份含量 50
表二、在20℃下自體酵素作用後柳橙精油中成份含量 51
表三、在30℃下自體酵素作用後柳橙精油中成份含量 52
表四、在50℃下自體酵素作用後柳橙精油中成份含量 53
表五、不同濃度之新鮮柳橙精油對Malassezia furfur生菌數之影響 54
表六、經30℃、7天自體酵素催化之不同濃度柳橙精油對Malassezia furfur生菌數之影響 55
表七、不同濃度經添加Mg2+在30℃下自體酵素作用三天之柳橙精油對Malassezia furfur生菌數之影響 56
表八、不同濃度之水溶性幾丁聚醣對Malassezia furfur生菌數之影響 57
表九、添加水溶性幾丁聚醣於1.0%新鮮柳橙皮精油乳化物中對Malassezia furfur生菌數之影響 58
表十、添加水溶性幾丁聚醣於1.0%經添加Mg2+在30℃下自體酵素作用三天之柳橙精油乳化物中對Malassezia furfur生菌數之影響 59

圖目錄
圖一、植物體內類?烯類化合物的生成途徑。 60
圖二、Geraniol及linalool的生成途徑。 61
圖三、於20、30、50℃下自體酵素作用對limonene的含量變化。 62
圖四、於20、30、50℃下自體酵素作用對linalool的含量變化。 63
圖五、添加不同濃度之Mn2+金屬離子在30℃下自體酵素作用7天後， linalool的含量變化。 64
圖六、添加不同濃度之Mg2+金屬離子在30℃下自體酵素作用7天後， linalool的含量變化。 65
圖七、皮屑芽胞菌之生長曲線。 66
圖八、水溶性幾丁聚醣之層析圖譜。 67

王以雯。2004。幾丁聚醣麥芽糖衍生物之製備、溶解性及保水性之探討。國立臺灣海洋大學食品科學系碩士學位論文。基隆，臺灣。
行政院農委會。2007。農業統計年報。臺北，臺灣。
李明遠。2000。醋酸乙醯化法製備水溶性幾丁聚醣之探討。國立臺灣海洋大學食品科學系碩士學位論文。基隆，臺灣。
呂文雄。1995。台灣農家要覽農作篇。豐年社出版社。臺北，臺灣。19-30。
佐佐木 薰。2002。芳香療法。智慧大學出版有限公司。臺北，臺灣。19-21。
林雨欣。2005。文旦皮精油之成分組成及其乳化物對S. aureus及E. coli的抗菌活性之探討。國立臺灣海洋大學食品科學系碩士學位論文。基隆，臺灣。
林俊然。2007。文旦皮精油及水溶性幾丁聚醣對皮屑芽孢菌之抗菌活性。國立臺灣海洋大學食品科學系碩士學位論文。基隆，臺灣。
卓芷聿。2006。精油大全。大樹林出版社。臺北，臺灣。1-35。
許玉華，2001，皮膚保健小常識，協合文化有限公司。7-12。
溫佑君。2006。香氣與空間。商周出版社。臺北，臺灣。134-141。
蔡政芳、林文源、李錦楓。1993。不同去乙醯度幾丁聚醣抑菌作用及澀味。中華生質能源學會會誌。12: 74-88.
蔡博仁。2001。葡萄柚皮中自體酵素轉換d-limonene為linalool最適條件之探討。國立臺灣海洋大學食品科學系碩士學位論文。基隆，臺灣。
A. O. A. C. (Association of Official Analytical Chemists). 1997. In: Official Methods of Analysis. 16th ed. Ed. Horwitz, W., and Washington, D. C.
Ashbee, H. R., and Evans, E. G. V. 2002. Immunology of diseases associated with Malassezia species. Clinical Microbiology Review. 15(1): 21-57.
Bailey, P., Arrowsmith, C., Darling, K., Dexter, J., Eklund, J., Lane, A., Little, C., Murray, B., Scott, A., Williams, A., and Wilson, D. 2003. A double-blind randomized vehicle-controlled clinical trial investigating the effect of ZnPTO dose on the scalp vs. antidandruff efficacy and antimycotic activity. International Journal of Cosmetic Science. 25: 183-188.
Basilico, M. Z., and Basilico, J. C. 1999. Inhibitory effects of some spice essential oils on Aspergillus ochraceus NRRL3174 growth and ochratoxin A production. Letters in Applied Microbiology. 29: 238-241.
Burt, S. A., and Reinders, R. D. 2003. Antibacterial activity of selected plant essential oils against essential oils against Escherichia coli O157:H7. Letters in Applied Microbiology. 36: 162-167.
Carson, C. F., Hammer, K. A., and Riley, T. V. 2006. Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clinical Microbiology Review. 19(1): 50-62.
Chang,C. P., and Dobashi, T. 2003. Preparation of alginate complex capsules containing eucalyptus essential oil and its controlled release. Colloids and Surfaces B:Biointerfaces. 32: 257-262.
Cheng, A. X., Lou, Y. G., Mao, Y. B., Lu, S., Wang, L. J., and Chen, X. Y. 2007. Plant terpenoids: biosynthesis and ecological functions. Journal of Integrative Plant Biology. 49(2): 179-186.
Deangelis, Y. M., Gemmer, C. M., Kaczvinsky, J. R., Kenneally, D. C., Schwartz, J. R., and Dawson, T. L. 2005. Three etiologic facets of dandruff and seborrheic dermatitis:Malassezia fungi, sebaceous lipids, and individual sensitivity. Journal Investigative Dermatology Symposium Proceedings. 10: 295-297.
Demyttenaere, J. C. R., Belleghem, K. V., and Kimpe, N. D. 2001. Biotransformation of (R) - (+) – and (S) - (-) – limonene by fungi and the use of solid phase microextraction for screening. Phytochemistry. 57: 199-208.
Douglas, J. O., Connor, C. H., and Thomas, E. M. 1998. Dietary chitosan inhibits hypercholesterolaemia and atherogenesis in the apolipoprotein E-deficient mouse model of atherosclerosis. Atherosclerosis. 138: 329-334.
Draelos, Z. D., Kenneally, D. C., Hodges, L. T., Billhimer, W., Copas, M., and Margraf, C. 2005. A comparison of hair quality and cosmetic acceptance following the use of two anti-dandruff shampoos. Journal Investigative Dermatology Symposium Proceedings. 10: 201-204.
Gershenzon, J., McConkey, M. E., and Croteau, R. B. 2000. Regulation of monoterpene accumulation in leaves of peppermint. Plant Physiology. 122: 205-213.
Hammer, K. A., Carson, C. F., and Riley, T. V. 2000. In vitro activities of ketoconazole, econazole, miconazole, and Melaleuca alternifolia (tea tree) oil against Malassezia species. Antimicrobial Agents and Chemotherapy. 44(2): 467-469.
Helander, I. M., Nurmiaho-Lassila, E. L., Ahvensinen, R., Rhoades, J., and Roller, S. 2001. Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. International Journal of Food Microbiology. 71: 235-244.
Herasa, A., Rodriguez, N., Ramos, V., and Agullo, E. 2001. N-methylene phosphonic chitosan: A novel soluble derivative. Carbohydrate Polymers. 44(1): 1-8.
Iscan, G., Kirimer, N., Kurkcuoglu, M., Baser, K. H. C., and Demirci, F. 2002. Antimicrobial screening of Mentha piperita essential oils. Journal of Agricultural and Food Chemistry. 50: 3943-3946.
Jia, Z., and Shen, D. 2002. Effect of reaction temperature and reaction time on the preparation of low-molecular-weight chitosan using phosphoric acid. Carbohydrate Polymers. 49: 393-396.
Kalemba, D. and Kunicka, A. 2003. Antibacterial and antifungal properties of essential oil. Current Medicinal Chemistry. 10(10): 813-829.
Kanatt, S. R., Chander, R., and Sharma, A. 2008. Chitosan and mint mixture: A new preservative for meat and meat products. Food Chemistry. 107: 845-852.
Kuzuyama, T. 2002. Mevalonate and nonmevalonate pathways for the biosynthesis of isoprene units. Bioscience, biotechnology, and biochemistry. 66(8): 1619-1627.
Landmann, C., Fink, B., Festner, M., Dregus, M., Engel, K. H., and Schwab, W. 2007. Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia). Archives of Biochemistry and Biophysics. 465(2): 417-429
Lee, M. H., Liu, H., Su, N. W., Ku, K. L., and Choong, Y. M. 1999a. Bicconversion of d-limonene to oxygenated compounds by endogenous enzymes of the citrus peel. Journal of the Chinese Agricultural Chemical Society. 37(1): 1-19.
Lee, M. H., Liu, H., Su, N. W., Wang, M. L., and Choong, Y. M. 1999b. Fractionation of endogenous enzymes reacted citrus peel oil by liquid/liquid extraction. Journal of the Chinese Agricultural Chemical Society. 37(1): 63-76.
Maeda, Y., and Kimura, Y. 2004. Antitumor effects of various low-molecular-weight chitosans are due to increased natural killer activity of intestinal intraepithelial lymphocytes in sarcoma 180-bearing mice. American Society for Nutritional Sciences. 134: 945-950.
Marostica Jr., M. R., and Pastore, G. M. 2007. Production of R-(+)-α-terpineol by the biotransformation of limonene from orange essential oil, using cassava waste water as medium. Food Chemistry. 101: 345-350.
McGarvey, D. J., and Croteau, R. 1995. Terpenoid Metabolism. The Plant Cell. 7: 1015-1026.
Minagawa, T., Okamura, Y., Shigemasa, Y., Minami, S., and Okamoto, Y. 2007. Effects of molecular weight and deacetylation degree of chitin/chitosan on wound healing. Carbohydrate Polymers. 67: 640-644.
Minh Tu, N. T., Thanh, L. X., Une, A., Ukeda, H., and Sawamura, M. 2002. Volatile constituents of Vietnamese pummelo, orange, tangerine and lime peel oils. Flavour and Fragrance Journal. 17: 169-174.
Mizuno, S., Kato, K., Ono, Y., Yano, K., Kurosaka, H., Takahashi, A., Abeta, H., Kushiro, T., Miyamoto, S., Kurihara, R., Hiki, N., Kaminishi, M., Iwasaki, A., and Arakawa, Y. 2006. Oral peppermint oil is a useful antispasmodic for double-contrast barium meal examination. Journal of Gastroenterology and Hepatology. 21: 1297-1301.
Nostro, A., Cannatelli, M. A., Morelli, I., Musolino, A. D., Scuderi, F., Pizzimenti, F., and Alonzo, V. 2004. Efficiency of Calamintha officinalis essential oil as preservative in two topical product types. Journal of Applied Microbiology. 97: 395-401.
Orav, A., and Kann, J. 2001. Determination of peppermint and orange aroma compounds in food and beverages. Proceedings of the Estonian Academy of Science Chemistry. 50(4): 217-225.
Oswald, M., Fischer, M., Dirninger, N., and Karst, F. 2007. Monoterpenoid biosynthesis in Saccharomyces cerevisiae. Federation of European Microbiological Societies. 7: 413-421.
Pichersky, E., Lewinsohn, E., and Croteau, R. 1995. Purification and characterization of S-linalool synthase, an enzyme involved in the production of floral scent in Clarkia breweri. Archives of Biochemistry and Biophysics. 316(2): 803-807.
Qin, C., Li, H., Xiao, Q., Liu, Y., Zhu, J., and Du, Y. 2006. Water-solubility of chitosan and its antimicrobial activity. Carbohydrate Polymers. 63: 367-374.
Qin, C., Du, Y., Xiao, L., Li, Z., and Gao, X. 2002. Enzymic preparation of water-soluble chitosan and their antitumor activity. International Journal of Biological Macromolecules. 31: 111-117.
Ran, Y., Yoshiike, T., and Ogawa, H. 1993. Lipase of Malassezia furfur: some properties and their relationship to cell growth. Journal of Medical and Veterinary Mycology. 31: 77-85.
Rensburg, E., Moleleki, N., Walt, J. P., Botes, P. J., and Dyk, M. S. 1997. Biotransformation of (+)limonene and (-)piperitone by yeasts and yeast-like fungi. Biotechnology Letters. 19(8): 779-782.
Rinaudo, M. 2006. Chitin and chitosan: Properties and applications. Progress in Polymer Science. 31: 603-632.
Ro, B. I., and Dawson, T. L. 2005. The role of sebaceous gland activity and scalp microfloral metabolism in the etiology of seborrheic dermatitis and dandruff. Journal Investigative Dermatology Symposium Proceedings. 10: 194-197.
Sanfilippo,A. and Joseph, C. 2006. An overview of medicated shampoos used in dandruff treatment. P&T. 31(7): 396-400.
Satchell, A. C., Saurajen, A., Bell, C., and Barnetson, StC. R. 2002. Treatment of dandruff with 5% tea tree oil shampoo. Journal of the American Academy of Dermatology. 47(6): 852-855.
Schelz, Z., Molnar, J., and Hohmann, J. 2006. Antimicrobial and antiplasmid activities of essential oils. Fitoterapia. 77: 279-285.
Shimahara, K., Ohkouchi, K., and Ikeda, M. 1982. A new isolation method of crustacean chitin using a proteolytic bacterium Pseudomonas maltophilia. In: Chitin and Chitosan. Proceedings of the Second International Conference on Chitin and Chitosan. The Japanese Society of Chitin and Chitosan Publishers. Ed. Hirano, S. and Tokura, S. Tottori. pp. 10-14.
Sreenath, H. K., Crandall, P. G., and Baker, R. A. 1995. Utilization of citrus by-products and wastes as beverage cluding agents. Journal of Fermentation and Bioengineering. 80: 190-194.
Verzera, A., Trozzi, A., Dugo, G., Di Bella, G., and Cotroneo, A. 2004. Biological lemon and sweet orange essential oil composition. Flavour and Fragrance Journal. 19: 544-548.
Zivanovic, S., Chi, S., and Draughon, A. F. 2005. Antimicrobial activity of chitosan films with essential oils. Journal of Food Science. 70: M45-M51.