臺灣博碩士論文加值系統

本研究擬開發酸凝酪及克弗爾兩種不同風味含有活菌的乳錠，並評估保護劑添加對乳錠原料中菌元凍乾後活性之影響。保護劑添加之最佳濃度及比例，是藉由反應曲面法設計（RSM）建立最佳方程式，再利用序列二次規劃法（SQP）獲得。酸凝酪乳錠製造方法是將脫脂乳添加益菌質後，再接種酸凝酪菌元(Lactobacillus bulgaricus, Streptococcus thermophilus) 及益生菌 （L. paracasei, Bifidobacterium longum,)，於37℃發酵12小時，混入抗凍保護劑，最後將樣品凍乾、壓錠製成風味獨特之益生菌酸凝酪乳錠。最佳化結果推薦，添加 6.98％半乳糖、7％蔗糖、8％脫脂乳粉為抗凍保護劑時，可得最佳的凍乾菌數存活率。在克弗爾酸凝酪製造方面，將脫脂乳添加5％的克弗爾粒，於20℃發酵24小時，再進行乾燥、打錠。最適化結果顯示，添加4.53％的半乳糖、7％蔗糖及5.03％的脫脂乳粉，可以得到最好的乳酸菌存活率，兩個產品的驗證實驗證實乳酸菌試驗結果與預測值間沒有顯著差異（P>0.05）。儲存結果顯示室溫下儲存6個星期，益生菌含量仍有105-107CFU/g；官能評估之結果發現雖然酸凝酪乳錠及克弗爾乳錠在外觀及質地上並無顯著的差異，而風味方面，克弗爾乳錠則有較低的評分。

A number of novel fermented dairy products are being developed and marketed under the concept of probiotic products, but few of probiotics were associated with confectionary goods. Thus, the objective of this research was to develop two probiotic candies (yogurt flavor and kefir flavor) by freeze drying and evaluate the effect of lyoprotectants on viability of probiotics. The proportion of lyoprotectants (galactose, skim milk power and sucrose) was optimized using response surface methodology (RSM) to first construct a surface model, with sequential quadratic programming (SQP) subsequently adopted to optimize the model. The endpoint was to increase the varieties of probiotic products and enhance their market values.
For manufacturing the yogurt flavor candy, skim milk was mixed with prebiotics and inoculated with yogurt cultures (Lactobacillus bulgaricus, Streptococcus thermophilus) and probiotics (L. paracasei, Bifidobacterium longum). After fermentation at 37℃ for 12 hours, the fermented samples were mixed with lyoprotectants, dehydrated by freeze drying and pressed to form tablets. Optimization results indicated that combination of 6.89% galactose, 7% sucrose and 8% skim milk powder would produce the highest survival in terms of probiotic count. For manufacturing the kefir candy, the processing steps were similar to yogurt samples except for the preparation of the kefir culture. To prepare the culture, skim milk was mixed with 5% kefir grains and incubated at 20℃ for 24 hours to produce the kefir culture. Results indicated that combination of 4.53% galactose, 7% sucrose and 5.03% skim milk powder would generate the highest survival of lactic acid bacterial count. The verification experiments yielded results close to the predicted values, with no significant difference (P>0.05) for both samples. The storage results demonstrated that these probiotic counts remained above 105-107 CFU/g stored for six weeks under room temperature. Sensory evaluation results revealed no significant difference between the yogurt and kefir samples in appearance and texture. However, the kefir flavor candy was sour and had the lower score in aroma.

壹、序言..................................................9
貳、文獻探討.............................................10
一、益生菌在台灣食品上之應用.............................10
二、凍乾 (freeze drying) 之應用..........................11
（一）凍乾法的原理及基本程序之介紹.......................12
（二）凍乾之優缺點.......................................15
三、凍乾於乳酸菌產品之利用...............................21
（一）乾燥益生菌之製備...................................21
（二）影響乾燥微生物存活之因子...........................21
（三）保護劑添加對乳酸菌存活率之影響.....................24
（四）保護劑添加對乳酸菌保存之影響.......................28
（五）儲存條件對乳酸菌保存之關係.........................29
四、錠劑之製造...........................................32
（一）錠劑製造過程變化...................................33
（二）錠劑添加物.........................................34
五、實驗設計.............................................38
（一）反應曲面法.........................................38
（二）序列二次規劃法.....................................39
參、材料與方法...........................................41
一、材料.................................................41
(一)樣品組成基質及保護劑................................41
(二)菌種及培養基........................................41
(三)試藥................................................42
(四)儀器設備............................................43
二、實驗方法.............................................47
（一）乳酸菌菌元及克弗爾菌元之保存.......................47
（二）探討益生菌乳錠最佳生產模式之實驗設計...............47
（三）微生物活性測定項目及分析方法.......................50
（四）益生菌乳錠物性測定項目及分析方法...................51
（五）品評實驗及儲存實驗.................................53
（六）統計分析...........................................54
肆、結果與討論...........................................59
一、酸凝酪菌元於牛乳中之發酵特性.........................59
二、酸凝酪乳錠原料最佳生產條件...........................65
（一）保護劑添加對凍乾酸凝酪菌數之影響...................65
（二）利用反應曲面法及序列二次規劃法探討酸凝酪乳錠原料最佳生產條件...................................................66
三、克弗爾乳錠原料最佳生產條件...........................76
（一）保護劑添加對凍乾克弗爾菌數之影響...................76
（二）利用反應曲面法及序列二次規劃法探討克弗爾乳錠原料最佳生產條件探討...............................................77
四、益生菌乳錠物性、化學分析及品評試驗之比較.............87
（一）酸凝酪乳錠及克弗爾乳錠物性分析.....................87
（二）酸凝酪乳錠及克弗爾乳錠化學成分分析.................87
（三）品評試驗之比較.....................................88
五、益生菌乳錠儲存在不同包材及溫度條件對菌數及水分之影響.......................................................88
伍、結論................................................101
陸、參考文獻............................................103

王金茂總編。1995。中華藥典。第四版。pp.65-66。台北市行政院衛生署。台灣。
王朝陽。2002。四逆湯錠劑之製劑學研究。中國醫藥學院中國藥學研究所。碩士論文。
王宜捷。2004。乳酸俊與雙叉桿菌發酵豆奶及其產品之抗氧化活性。台灣大學食品科技研究所。碩士論文。
邱欣怡。2004。藉最適化方法探討添加益菌質於囊壁材質對為膠囊化原生菌之影響。台灣大學畜產研究所。碩士論文。
汪昭期。2004。發酵條件和保護劑對Lactobacillus reuteri Pg 4保存之探討。中興大學畜產學研究所。碩士論文。
吳俊彥。2003。半纖維素水解液中木糖分離及其發酵。大業大學食品工程學所。碩士論文。
林慶文。1993。乳製品之特性與機能性。華香園出版社，台北。
林世偉。2003 凍乾對乳酸菌存活率與其主要生理特性之影響。東海大學畜產學研究所。碩士論文。
林志宏。2004藉由改變乳酸菌代謝途徑的方式增加山梨醇產量。清華大學生物科技研究所。碩士論文。
柯紀庸。2002。製錠條件對DiltiazemHcl錠劑之溶離效應暨體內吸收之影響。高雄醫學大學藥學研究所。碩士論文。
洪肇宏。2004。天麻錠劑之配方研究。中國醫藥大學藥物化學研究所碩士論文。
周佳蓉。2003。藉由遺傳演算法與序列二次規劃法探討原生菌奶豆腐最佳製造條件。台灣大學畜產研究所。碩士論文。
連紋乾。2000。保健膳食補助品之備製-利用阿拉伯膠及脫脂乳粉低溫噴乾為膠囊化雙差桿菌。台灣大學食品科技研究所。碩士論文。
許佳鳳。2005。克弗爾於皮膚特性影響之研究。台灣大學畜產學研究所。碩士論文。
郭孟鎧。2001。Oxatomide錠劑配方設計及研究。中國醫藥學院醫藥化學所。碩士論文。
葉又慈。2003。羊乳克弗爾的微生物生長特性極其特殊脂肪酸之研究。台灣大學畜產學研究所。碩士論文。
賴滋漢、金安兒、柯文慶。1993。食品加工學（方法篇）。精華出版社，台中。
黃俊達。1995。不同乳酸菌應用於乾燥酸酪乳製造之研究。東海大學畜產學研究所。碩士論文。
黃保林。1989。基礎藥劑學pp.351-367。睿煜出版社。台灣。
黃鎮富。1999。機能性軟糖中食用膠添加配方與質地相關性之研究。大業大學食品工程學系研究所。碩士論文。
蘇家俊。2001。不同乾燥方法對添加各種保護劑之L. casei CCRC 14023菌株儲藏效果探討。中興大學食品科學系。碩士論文。
Abadias, M., A. Benabarre, N. Teixido, J. Usall and I. Vinas. 2001. Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake. Int. J. Food Microbiol. 65:173-182.
Arunachalam, K. D. 1999. Role of bifidobacteria in nutrition, medicine and technology. Nutr. Res. 19:1559-1597.
Berny, J. F. and G. L. Hennebert. 1991. Viability and stability of yeast cells and filamentous fungus spores during freeze drying: Effects of protectants and cooling rates. Mycologia. 83: 805-815.
Box, G. E. P. and D. W. Behnken. 1960. some new three levele designs for the study of quantitative variables. Technometrics 2:455-475.
Bozoglu, T. F., M. Ozilgen and V. Bakir. 1987. Survival kinetics of lactic acid starter cultures during and after freeze-drying. Enzyme Microb. Technol. 9:531-537.
Brennan, M., B. Wanismail, M. C. Johnson and B. Ray. 1986. Cellular damage in dried Lactobacillus acidophilus. J. Food Prot. 49:47-53.
Bullimore B. K. 1983. Dairy starters reviewed. Dairy Ind. Int. 48:19-23.
Burt, B. A. 2006. The use of sorbitol- and xylitol-sweetened chewing gum in caries control. J. Am. Dent. Assoc.137(2): 190 - 196.
Capela, P., T. K. C. Hay and N. P. Shah. 2006. Effect of cryoprotectants, prebiotics and microencapsulation on survival of probiotic organism in yoghurt and freeze-dried yoghurt. Food Res. Int. 39: 203-201
Carvalho, A. S., J. Siva, P. Ho, P. Teixeira, F. X. Malcata and P. Gibbs. 2004. Relevant factors for the preparation of freeze-dried lactic acid bacteria. Int. Dairy J. 14: 835-847.
Carvalho, A. S., J. Silva, P. Ho, P. Teixerira, F. X. Malcata and P. Gibbs. 2002. Effect of additives on survival of freeze-dried Lactobacillus plantarum and Lactobacillus rhamnosus during storage. Biotechnol. Lett. 24:1587-1591.
Castilla, O. S., C. L. Calleros, E. A. Mandujano and E. J. V. Carter. 2004. Microstructure and texture of yogurt as influenced by fat replacers. Int. Dairy J. 14: 151-159.
Castro, H. P., P. M. Teixeira and R. Kirby. 1995. Storage of lyophilized cultures of Lactobacillus bulgaricus under different relative humidities and atmospheres. Appl. Microbiol. Biotechnol. 44(1,2): 172–176.
Champagne, C. P., N. J. Gardner, E. Brochu and Y. Beaulieu. 1991. The freeze-drying of lactic acid bacteria. A review. Food Sci. Technol. J. 24: 118-128.
Champagne, C. P., F. Mondou, Y. Raymond and D. Roy. 1996. Effect of polymers and storage temperature on the stability of freeze-dried lactic acid bacteria. Food Res. Int. 29: 555-562.
Champagne, C. P. and N. J. Gardner. 2001.The effect of protective ingredients on the survival of immobilized cells of Streptococcus thermophilus to air and freeze-drying. Electronic J. Biotechnol. 4: 146-152
Chen, H. C., C. W. Lin and M. J. Chen. 2005. The effect of freeze drying and rehydration on survival of microorganisms in kefir. Asian-Australas. J. Anim. Sci. 19(1): 126-130.
Collins, J. K., G. Thornton and G. O. Sullivan. 1998. Selection of probiotic strains for human applications. Int. Dairy J. 8: 487-490.
Converti, A., P. Perego, A. Sordi and P. Torre. 2002. Effect of starting xylose concentration on the microaerobic metabolism of Debaryomyces hansenii. The use of carbon material balances. Appl. biochem. Biotechnol. 101(1): 15–29.
Costa, E., J. Usall, N. Teixido, N. Garcia, and I. Vinas. 2000. Effect of protective agents, rehydration medium and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying. J. Appl. Microbiol. 89: 793-800.
Costa, E., J. Usall, N. Teixido, R. Torres and I. Vinas. 2002. Effect of package and storage conditions on viability and efficacy of the freeze-dried biocontrol agent pontoea agglomerans strain CPA-2. J. Appl. Microbiol. 92: 873-878.
Fellows, P. 2000. Food Processing Technology : principles and practice. Chichester, England. USA. 22: 441-452.
Font, de Valdez, G., G. S. de Giori, A. P. de Ruiz Holgado and G. Oliver. 1983. Comparative study of the efficiency of some additives in protecting lactic acid bacteria against freeze-drying. Cryobiology. 20: 560-566.
Fratzke, A. R. and P. J. Reilly, 1977. Uses and Metabolic Effects of Xylitol, I. Process Biochem. 12(7) : 27-29.
Fry, R. M. 1966. Freezing and drying of bacteria. In: Meryman H. T. ed. Cryobiology. London : Academic Press, 665-696.
Gilliland, S. E. and H. S. Kim. 1984. Effect of viable starter culture bacteria in yoghrt on lactose utilization in humans. J. Dairy Sci. 67:1-6.
Gismondo, M. R., L. Drago and A. Lombardi. 1999. Review of probiotics available to modify gastrointestinal flora. Int. J. Antimicrob. Agents. 12: 287-292.
Goldberg, I. and L. Eschar. 1977. Stability of lactic aicd bacteria to freezing as related to their fatty acid composition. Appl. Environ. Microbiol. 33:489-496.
Gomes, A. M. P. and F. X. Malcata. 1999. Bifidobacterium spp. and Lactobacillus acidophilus: biological, biochemical, technological and therapeutical properties relevant for use as probiotics. Trend Food Sci. Technol. 1:139-157.
Gorp, K.V., E. Boerman, C. V. Cavenaghi and P. H. Berben. 1999 Catalytic hydrogenation of then fine chemical:sorbitol production. Lsevier. 349-361
Grill, J. P., C. Cayuela, J. M. Antonie and F. Schneider 2000. Effects of Lactobacillus amylovorus and Bifidobacterium breve on cholesterol. Lett. Appl. Microbiol. 31:154-156.
Hanafusa, J. V. 1985. The hydration water and protein with cryoprotectant. In: Fundamentals and applications of freeze-drying to biological materials, drugs and food stuffs. International Institute of Refrigeration. Paris. pp. 59.
Hirayama, K. and J. Rafter. 2000. The role of probiotic bacteria in cancer prevention. Microb. Infec. 2: 681-686.
Hirota, T. 1990. The anticipating bioactivity functions of Lactobacillus in dairy products. New Food Ind. 32: 9-17.
Hose, H. and T. Sozzi 1991. Biotechnology group meeting probiotics fact or fiction. J. Chem. Technol. Biotechnol. 51: 540-544.
Hsiao, H. C., Lian W. and C. C. Chou 2002. Viability of various microencapsulated bifidobacteria during storage. Xth International Congress of Bacteriology and Applied Microbiology Microbial World, July 27-Aug 1, 2002. Paris, France.
Ishibashi, N. and Shimamura S. 1993. Bifidobacteria: Research and development in Japan. Food Technol. 47:126-134.
Jiang, T., A. Mustapha and D. A. Savaiano. 1996. Improvement of lactose digestion in humans by injection of fermented milk containing Bifidobacterium longum. J. Dairy Sci. 79:750-757.
Johnson, J. A. C. and M. R. Etzel. 1995. Properties of Lactobacillus helveticus CNRZ-32 attenuated by spray-drying, freeze- drying, or freezing. J. Dairy Sci. 78: 761-768.
Kasper, H. 1998. Protection against gastrointestinal disease - present facts and future developments. Int. J. Food Microbiol. 41:127-131.
Kearney, L., M. Upton and A. McLaughlin. 1990. Enhancing the viability of Lactobacillus plantarum inoculum by immobilizing cells in calcium alginate beads incorporating cryoprotectants. Appl. Environ. Microbiol. 56: 3112-3116.
Koca, N. and M. Metin. 2004. Textural, melting and sensory properties of low-fat fresh kashar cheeses produced by using fat replacers Int. Dairy J. 14 : 365–373.
King, V. A. E., and J. T. Su. 1993. Dehydration of Lactobacillus acidophilus. Proc. Biochem. 28: 47-52.
Marteau, P. R., M. Vrese, C. J. Cellier and J. Schrezenmeir. 2001. Protection from gastrointestinal disease with the use of probiotics. Am. J. Clin. Nutr. 73: 430-436.
Molder, H. W., R. C. McKeller and M. Yaguchi. 1990. Bifidobacteria and bifidogenic factors. Inst. Food Sci. Technol. J. 23: 29-41.
Parajo, J. C., H. Domınguez and J. M. Domınguez, 1998. Biotechnological production of xylitol. Part 1: Interest of xylitol and fundamentals of its biosynthesis. Bioresource Technol. 65(3), 191–201.
Sampaio, F. C., C. A. Moraes, D. D. Averi , P. Perego, A. Converti and F. M. L. Passosa. 2006 Influence of temperature and pH on xylitol production from xylose by Debaryomyces hanseni. Process Biochem. 41: 675-681.
Speckman, C. A., W. E. Sandine and P. R. Elliker. 1974. Lyophilized lactic acid starter culture concentrates: preparation and inoculation of vat milk for cheddar and cottage cheese. J. Dairy Sci. 57: 165-175.
Szoke, J., J. Banoczy and H. M. Proskin 2001 Effect of after-meal sucrose-free gum-chewing on clinical caries J. Dent. Res. 80： 1725-1729.
Tamime, A. Y. and R. K. Robinson. 1985. Yoghurt : science and technology. 1st ed. New York: Oxford.
Tannock, G. W. 1997. Probiotic properties of lactic-acid bacteria: plenty of scope for fundamental R and D. Tibech 15: 270-274.
Teixeria, P., H. Castro and R. Kirby. 1995. Spray drying as a method for preparing concentrated cultures of Lactobacillus bulgaricus. J. Appl. Bacteriol. 78:456-462.
Van Arsdel, W. B., M. J. Copley and Morgan A. I. 1973. Food dehydration. 2nd ed. Westport, Conn.: Avi Pub. Co.
Veen, V. B., K. V. Maarschalk, G. K. Bolhuis, K. Zuurman, and H W.Fijlink. 2000. Tensile of tablets containing two materials with a different compaction behaviour. Int. J. Pharm. 203:71-79.
Wang, Y. C., R. C. Yu and C. C. Chou. 2003. Viability of lactic acid bacteria and bifidobacteria in fermented soymilk after drying, subsequent rehydration and storage. Int. J. Food Microbiol. 93:209-217.
Washuttle, J., P. Riederer and E. Banchem. 1973. A qualitative and quantitative study of sugar-alcohols in several food. J. Food Sci. 38: 26-35.
Wollowski, I., G. Rechkemmer and B. L. Pool-Zobel. 2001. Protective role of probiotics and prebiotics in colon cancer. Am. J. Clin. Nutr. 73: 451-455.
Yamagata. 1965. Clinical effect of xylitol on carbohydrate and lipid metabolism in diabetes. The Lancet. 2 (7419) : 918-921.
Yazici, F. and A. Akgun. 2004. Effect of some protein based fat replacer on physical, chemical, textural, and sensory properties of strained yogurt. J. Food Eng. 62: 245-254.
Zayed, G. and Y. H. Roos. 2004. Influence of trehalose and moisture content on survival of Lactobacillus salivarius subjected to freeze-drying and storage. Process Biochem. 39:1081-1086.
Zhao, G. and G. Zhang. 2005. Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying. J. Appl. Chem. Biotechnol. 99:333-338.