臺灣博碩士論文加值系統

本研究分為二部分：(1) 乳酸菌分離與鑑定；(2) 乳酸菌刺五加發酵保健飲料之開
發。我們由市面上味全LCA506 優酪乳分離、純化與API 生化分析與PCR 分子鑑定，得
到乳酸菌為一Lactobacillus paracasei subsp. paracasei strain FQ005 (LPSPS5)。LPSPS5 乳
酸菌與刺五加及水分別加入不同量的蔗糖(0%、5%、10%、15%、20%)或脫脂乳(0%、
1%、2%、3%、4%)下於室溫下發酵培養。發現於含5%蔗糖或3%脫脂乳的發酵刺五加
液有最大的乳酸菌數，分別為4.7*108 cells/mL 與1*109 cells/mL。以雙因子的中央合成
設計法(central composite design, CCD)與實驗設計軟體(Design Expert)進行碳源
(5%蔗糖)與氮源(3%脫脂乳)之最佳配方推算與實驗驗證，5%蔗糖與3%脫脂乳可生成
最佳乳酸菌數目。我們將乳酸菌、5%蔗糖和3%脫脂乳與刺五加液進行發酵，於86 小
時發酵期間，我們發現LPSPS5 乳酸菌生長於36 小時與72 小時有最佳生長數目，其生
長情形呈現雙波峰之現象；其pH 由5.5 下降至3.4；乳酸菌刺五加發酵液，發現都有很
顯著量的總多酚，介於120 至220 mg Gallic acid/mL；總糖量介於6.0 至2.4 mg glucose/mL；
有很顯著的DPPH 自由基清除力，介於4.7 至5.5 mg Gallic acid/mL；很顯著的澱粉酶活
性，介於5.0 至27.0 unit/mL；以氣相層析儀分析(GC)沒有發現甲醇、乙醇與乙酸含量；
乳酸菌刺五加發酵液於風味、酸度、甜度、澀度、整體感、總接受度有很好的評價。
綜其上述，乳酸菌刺五加發酵液有潛力成為一維護健康之保健飲料。

This study is divided into two parts: (1) Isolation and identification of lactic acid bacteria; (2)
Development of fermented health beverage of Acanthopanax by lactic acid bacteria. A lactic
acid bacterium was isolated, purified and identified from Wei Chuan LCA506 yogurt
obtained from market and further identified as Lactobacillus paracasei subsp. Paracasei strain
FQ005 (LPSPS5) by API biochemical analysis and PCR molecular identification. The
LPSPS5 lactic acid bacteria, Acanthopanax, water with different amounts of sucrose (0%, 5%,
10%, 15%, 20%) or skimmed milk (0%, 1%, 2%, 3%, 4%) were mixed together and cultured
at room temperature. The fermented Acanthopanax broth containing 3% sucrose or 5% skim
milk has been found to produce maximum number of lactic acid bacteria, 4.7*108 cells/mL
and 1*109 cells/mL, respectively. The best formula was selected as 5% sucrose and 3% skim
milk by experimental identification of the best formula composition of lactic acid bacteria
growth predicted by central composite design. The LPSPS5 lactic acid bacteria, 5% sucrose
and 3% skim milk, and Acanthopanax liquid were added together and fermented. During 86
hr’s fermentation, the growth curve of LPSPS5 lactic acid bacteria has shown a double peak
that had the highest bacteria number at 36 and 72 hour. The pH was decreased from 5.5 to 3.4;
The Acanthopanax fermentation broth were found to have a significant amount of total
polyphenols, between 120 and 220 mg gallic acid / mL; The total sugar contents were ranged
from 6.0 to 2.4 mg glucose / mL; There are significant DPPH radical scavenging activity,
between 4.7 and 5.5 mg Gallic acid/ mL; The amylase activity was significantly existed with
5.0 to 27.0 unit / mL; There is no detectable amount of methanol, ethanol and acetic acid in
fermented broth by GC analysis. There are good scores of flavor, acidity, sweetness,
astringent degree, the overall sense, and the total acceptance in sensory evaluation of
Acanthopanax fermentation broth. Taken together, the Acanthopanax fermentation broth by
lactic acid bacteria has potential to be a health care beverage.

摘要 ................................................................................................................................. 1
Abstract ........................................................................................................................... 2
誌謝 ................................................................................................................................. 3
目錄 ................................................................................................................................. 4
第一章 前言 ................................................................................................................... 7
第二章 文獻回顧........................................................................................................... 9
2.1.1 益生菌簡介 ................................................................................................. 9
2.1.2 乳酸菌定義 ................................................................................................. 9
2.1.3 乳酸菌之分類 .......................................................................................... 10
2.1.4 乳酸菌益處 .............................................................................................. 10
2.2.1 刺五加介紹 .............................................................................................. 12
2.2.2 刺五加的由來及原性狀 ......................................................................... 13
2.2.3 刺五加的主要產地分布 ......................................................................... 13
2.2.4 刺五加於傳統醫學中的功效 ................................................................. 13
2.2.5 刺五加臨床應用 ..................................................................................... 14
2.2.6 刺五加化學成分 ..................................................................................... 16
第三章 實驗材料與方法 ............................................................................................ 18
3.1 實驗材料 ..................................................................................................... 18
3.1.1 使用藥品 ........................................................................................ 18
3.1.2 使用儀器 ........................................................................................ 19
3.2 實驗方法 ..................................................................................................... 20
3.2.1 乳酸菌培養、分離與鑑定 20
3.2.2 乳酸菌活化 20
3.2.3 液態培養 21
3.2.4 乳酸菌鑑定 21
5
3.2.4.1 API 生化鑑定分析 21
3.2.4.2 PCR 分子生物鑑定 22
3.2.5 乳酸菌刺五加發酵液分析 23
3.2.5.1 刺五加萃取配置 23
3.2.5.2 乳酸菌於刺五加萃取發酵 24
3.2.5.3 乳酸菌數測定 24
3.2.5.4 實驗設計與分析方法 24
3.2.6 乳酸菌刺五加發酵液功能性分析 24
3.2.6.1 總多酚測定 24
3.2.6.2 DPPH 自由基清除能力測定 25
3.2.6.3 總糖測定 27
3.2.6.4 澱粉分解脢活性測定 27
3.2.6.5 GC 分析甲醇、乙醇或有機酸生成 29
3.2.6.6 感官品評 29
3.2.6.7 數據處理 30
第四章 結果與討論..................................................................................................... 31
4.1 乳酸菌培養、分離與鑑定 ......................................................................... 31
4.1.1 乳酸菌的分離、純化及保藏 31
4.1.2 API 生化分析鑑定 32
4.1.4 PCR 分子生物鑑定 32
4.2 LPSPS5 乳酸菌生長與發酵刺五加飲料之開發 ....................................... 36
4.2.1 不同碳源或氮源的添加對LPSPS5 乳酸菌生長之評估 36
4.2.1.1 LPSPS5 乳酸菌於含不同量蔗糖之刺五加生長 36
4.2.1.2 LPSPS5 乳酸菌於含不同量脫脂乳之刺五加生長 37
4.2.1.3 雙因子的中心組實驗設計推算最高生長數目之最佳配方與驗
證 37
4.2.2 最適配方之發酵 39
4.2.2.1 最適配方之刺五加發酵液乳酸菌生長情況與pH 值變化 39
4.2.2.2 最適配方發酵之乳酸菌刺五加發酵液之功能性分析 41
4.2.2.3 總多酚 41
4.2.2.4 總糖 42
4.2.2.5 DPPH 自由基清除力 43
4.2.2.6 澱粉酶活性測定 44
4.2.2.7 GC 分析甲醇、乙醇或有機物成量 45
4.2.2.8 感官品評 47
4.3 結論 ............................................................................................................. 48

林裕森.葡萄酒全書.台北市:宏觀文化.1997。
張永和.發酵與發酵食品.靜宜大學食品營養系。
徐華強.Hirschfelder:15.2009。
潘子明.我國乳酸菌最近的研究趨勢暨通過健康食品認證之乳酸菌產品現況.農業
生技產業季刊 3: 19-27. 2005.
陳慶源.綜論乳酸菌之多元化應用.食品工業40(9): 1-3. 2008.
郭婕.認識刺五加.輔仁大學食品營養研究所博士班。
歐明 （1999）。常用中藥手冊。台北市：旺文社。
祖元剛（2005）。刺五加生活史型特徵及其形成機制。北京市：科學出版社。
許筑維、王盛世。以酵母菌製備綠茶飲料之量產製程開發與分析。2011 綠環境技
術學術研討會論文集, p24-31，2011/11/24，(高苑科技大學)。國際標準書號
(ISBN)：978-986-6755-42-2
王盛世*、謝震、徐嘉澤、李岱紋。酵母菌發酵綠茶液過程中酶活性與糖的變化
探討。2009 綠環境技術學術研討會論文集, p19-25，2009/12/19，(高苑科技
大學)。國際標準書號(ISBN)：978-986-6755-42-2
許筑維、王盛世。不同碳源或氮源引響北蟲草於綠茶液生長及生長時期綠茶發酵
液之功能性能力變異之探討。高苑科技大學化工與生化工程系研究所碩士論
文。
徐向宏，何明珠.实验设计与 Design- Expert 、 SPSS 应用 [M].北京:科学出版社，
2010: 146 一 157.
中國醫學科學院藥物研究所(1981):中藥志(第一冊)。中國。
中國中醫研究院(1992):實用中醫辭典。台北。知音出版社。
中藥大辭典，第一冊。昭人出版社。台北。(1980)
久保德三(1993):刺五加的效用。台北:青春出版社。
53
久鄉晴彥、近藤嘉和(1991):刺五加的驚人療效。台北縣:正義出版社。
Alm, L., Ryl-Kjelln, E., Setterberg, G and Blomquist, L. 1993. Effect of a new fermented
milk product”sultura” on constipation in geriatric patients. The Lact Acid Bact
Comp Conf.1-4
Black, F.T.,Andersen, P.L., Qrskov, F., Gaarslev, K. and Laulund, S.1989. Prophylactic
efficacy of lactobacilli on travele’s diarrhea.Travel Medicine.3:333-335
Dambekodi P.C. and Gilliland S.E. 1998. Incorporation of cholesterol into the cellular
membrane of Bifidobacterium longum. J Dairy Sci 81:1818-1824
Folin O, Denis W: A colormetric method for the determination of phenols ( and
phenol derivates) in urine, J Bio Chem 1915, ⅩⅩⅡ, No 2.
Fukushima, Y.,Kawata, Y.,Hara H., Terada, A. and Mitsuoka, T .1998.Effect of probiotic
formula on intestinal immunoglobulim A production in healthy children. Int. J.
Food Microbiol.42:39-44
F. J. Carr, D. Chill and N. Maida, The lactic acid bacteria: a literature survey. Crit Rev
Microbiol 28: 281-370. 2002.
Gilliland, S.E., Bruce, B.B., Bush, L.J. and Staley, T.E. 1980.Comparison of two strains of
Lactobacillus acidophilus as dietary adjuncts for young calves. J.Dairy
Sci.63:964-972
Gilliland, S.E., Nelson, C.R., and Maxwell, C. 1985. Assimilation of cholesterol by
Lactobacillus acidophilus.appl. Environ. Microbiol.49:377-381
Gomes AMP, Malcata FX. 1999. Bifidobacterium spp. and Lactobacillus acidophilus:
Biological, biochemical, technological and therapeutical properties relevant for
use as probiotics. Trends Food Sci 10: 139-157.
Kaur, C. and H. C. Kapoor. 2001. Antioxidants in fruits and vegetables – the
millennium’s health. Int. J. Food Sci. Technol. 36: 703-725.
Klaver F.A., and van der Meer R. 1993.The assumed assimilation of cholesterol by
Lactobacilli and Bifidobacterium bifidum is dve to their bile salt-deconjugating
activity. Appl Environ Microbiol 59:1120-1124
Linbeck, A. and Nord, C. E. 1991. Lactobacilli in relation to human ecology and
antimicrobial therapy. Int J Tiss Reac 13:115-122
Link-Amster H., Rochat F., Saudan K. Y., Mignot O. and Aeschlimann J.M. 1994.
Modulation of a specific humoral immune response and changes in intestinal
flora mediated through fermented milk intake. FEMS Immunol Med Microbiol
10:55-63
Mann,G.V. and Spoerry, A. 1974. Studies of a surfactant and cholesteremia in the
Massai. Am J ClinNutr.27:464-469
54
M. Sarrela, L. Lahteenmaki, R. Crittenden, S. Salminen and T. Mattila-Sandholm, Gut
bacteria and health foods 3/4 the European perspective. Int J Food Microbiol 78:
99-117. 2002.
Naik, G. H., K. I. Priyadarsini, J. G. Satav, M. M. Banavalikar, D. P. Sohoni, M. K. Biyani
and H. Morhan. 2003. Comparative antioxidant activity of individual herbal
components used in Ayurvedic medicine. Phytochem. 63: 97-104.
Nord C.E., Lidbeck A., Orrhage K. and Sjostedt S. 1996. Oral supplementation with
lactic acid-producing bacteria during intake of clindamycin. Clinical
Microbiol.Infect.,3:124-132
Ruch, R. J., S. Cheng and J. E. Klaungig. 1989. Prevention of cytotoxicity and inhibition
of intercellular communication by antioxidant catechin isolated from Chinese
green tea. Carcinogensis. 10: 1003-1008.
R. Havennar and J. H. J. Husis in’t Veld, The Lactic Acid Bacteria. Wood B.J.B. ed.
1:151-170. 1992.
Shimada, K., K. Fujikawa, K. Yahara and T. Nakamura. 1992. Antioxidant properties of
xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric.
Food Chem. 40: 945-948.
Tahri K., Grill J.P. and Schneider F. 1997. Involvement of trihydrooxyconjugated bile
salts in cholesterol assimilation by bifidobacteria. Curr Microbiol 34:79-84