臺灣博碩士論文加值系統

近年來食品工業的發展，食品添加物中含有機能性的性質，像是增進腸胃道益生菌群以紓緩便秘、降低血中膽固醇和促進鐵離子吸收。現行的食品工業可使用不同微生物經由蔗醣生產出果寡醣。在許多研究中都有果寡醣的產出，而到目前為止只有少數的研究中有新果寡醣的產出。由於新果寡醣特定的支鏈鍵結，使其具有較優於果寡醣的化學性質(耐熱性、耐酸性)，也因高純度的新果寡醣熱量低，可供醣尿病患者食用，其商業價值遠高於一般傳統果寡醣。本研究是為製備高純度新果寡糖並加以定量，利用0.05N H2SO4硫酸水解新果寡糖（Neo-Fructooligosaccharides，通常簡寫作Neo-FOS）使其分解為新蔗果三糖（neokestose）和新蔗果四糖（neotetrasaccharide），再回推標準品以計算新果寡糖的量。

In recent years, the development of the food industry, food additives containing functional properties, such as the promotion of gastrointestinal probiotic group to relieve constipation, reduce blood cholesterol and promote iron absorption. The current food industry can use different sugar produced via microbial FOS. FOS has outputs in many studies, but so far only a few studies have outputs new FOS. As the new branched fructooligosaccharides specific binding, it has a better than FOS chemical properties (heat resistance, acid resistance), but also because the new high-purity low heat FOS available for diabetics, its commercial value is much higher than traditional FOS.The new study is the preparation of high purity FOS and be quantified using 0.05N H2SO4 sulfuric acid hydrolysis new FOS (Neo-Fructooligosaccharides, usually abbreviated as Neo-FOS) to break down the new kestose (neokestose) and new nystose (neotetrasaccharide), return to push the new standard to calculate the amount of FOS.

第一章 緒 論 1
1.1. 研究動機 1
1.2. 文獻回顧 2
1.2.1. 果寡醣簡介 2
1.2.2. 新果寡醣簡介 10
1.2.3. 益生菌簡介 13
1.2.4. 益生素 20
1.2.5. 果寡醣與益生菌之關係 20
1.2.6. 高效液相層析（High Performance Liquid Chromatography：HPLC） 22
1.3. 減壓濃縮機 26
1.3.1. 減壓濃縮機原理 26
1.4. 研究目的 26
第二章 材料與方法 27
1.1 新果寡醣 27
1.1.1酵母菌Xanthophyllomyces dendrorhous TU31 27
1.1.2用醱酵槽生產「高純度新果寡醣」 27
2.儀器設備 28
3.方法 28
3.1 標準品製備 28
3.2 新果寡醣收集 28
4.分析 31
5.定量法 31
6.利用標準品回推定量 31
7. 研究架構 32
第三章 結果與討論 33
1. 純化新果寡醣 33
2. 比較硫酸水解後有無冰浴 34
2.1新果蔗三醣硫酸水解之數值(未冰浴) 35
2.2.新果蔗三醣硫酸水解之數值(冰浴) 37
2.3.新果蔗四醣硫酸水解之數值(未冰浴) 39
2.4.新果蔗四硫酸水解之數值(冰浴) 41
2.5 有無冰浴之結果 43
3. 比較不同硫酸濃度水解 44
3.1硫酸水解新果蔗三醣XY分布圖(0.1N) 45
3.2硫酸水解新果蔗三醣XY分布圖(0.05N) 47
3.3硫酸水解新果蔗四醣XY分布圖(0.1N) 49
3.4硫酸水解新果蔗四醣XY分布圖(0.05N) 51
3.5不同硫酸濃度水解之結果 53
4. 標準品定量 54
4.1 面積換算濃度-新果蔗三醣 55
4.2 面積換算濃度-新果蔗四醣 56
4.3 面積換算濃度-結果 57
5. 討論 59
第四章 結論 60
參考文獻 61

1.Alvaro-Benito M, De Abreu M, Fernandez-Arrojo L, Plou F. J, Jimenez-Barbero J, Ballesteros A, Polaina J, Fernandez-Lobato M. 2007. Characterization of a betafructofuranosidase from Schwanniomyces occidentalis with transfructosylating activity yielding the prebiotic 6-kestose.J Biochem 132:75-8 1.

2.Ben Ammar Y, Matsubara T, Iizuka M, Limpaseni T, Ponsawasdi P. 2001. Some properties of levansucrase of Bacillus natto stabilized with periodate oxidized yeast glucomannan. Enzym Microb Technol 30:875–882.

3.Crittenden R. G,Playne M. J. 1996. Production,properties and applications of food grade oligosaccharides. Trends Food Sci Technol 7:353.

4.Dimitris C, Robert AR ed. 2009. Prebiotics and probiotics science and technology. ISBN: 978-0-387-79057-2. Springer

5.Duan K. J, Sheu D. C, Chen J. S. 1993. Purification and characterization of β-fructofuranosidase from Asp. japonicus TIT-KJ1. Biosci Biotech
Biochem 57:1811–1815.

6.El-Refai HA, Abdel-Fattah AF, Mostafa FA. 2009. Enzymic synthesis of levanand fructo-oligosaccharides by Bacillus circulans and improvement of levansucrase stability by carbohydrate coupling. World J Microbiol Biotechnol 25:821–827.

7.Fishbein L, kaplan H, Gough M.1988. Fructooligosaccharides : A review.Toxicology 30:104-107.

8.Gibson G. R, Willis C. L, van Loo J. 1994. Non-digestible oligosaccharides and bifidobacteria – implications for health. Int Sugar J 96, 381–387.

9.Gross M, Ceier G, Rudolph K, Cieder K. 1992. Levan and levansucrase synthesized by the fire blight pathogen Erwinia amylovora. Physiol Mol Plant Pathol 40:371–381.

10.Hang Y, Woodams E, Jang K. 1995. Enzymatic conversion of sucrose to kestose by fungal extracellular frucωsyltransferase. Biotechnol Lett 17:295-298.

11.Hayashi S, Yoshiyama T, Fujii N, Shinohara S. 2000. Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum. Biotechnol Lett 22, 1465–1469.


12.Hidaka H,Eida T,Takizawa T,Tokunaga T, Tashiro Y. 1986. Effects of fructooligosaccharides on intestinal flora and human health. Bifid Microflora 5:37-50.

13.Jing Chen, Xiaoming Chen, Xueming Xu, Yawei Ning, Zhengyu Jin, Yaoqi Tian. 2011. Biochemical characterization of an intracellular 6G-fructofuranosidase from Xanthophyllomyces dendrorhous and its use in production of neo-fructooligosaccharides (neo-FOSs). Biores Technol 102:1715–1721

14.Kaur N, Gupta A. K. 2002. Applications of inulin and oligofructose in health and nutrition. J. Biosci 27:703-714.

15.Kilian S, Kritzinger S, Rycroft C, Gibson G, du Preez J. 2002. The effects of the novel bifidogenic trisaccharide, neokestose, on the human colonic microbiota. World J Microbiol Biotechnol 18: 637–644.

16.Kilian S. G, Sutherland FCW, Meyer PS, du Preez JC. 1996. Transport-limited sucrose utilization and neokestose production by Phaffia rhodozyma. Biotechnol Lett 18:975–980.

17.Kritzinger S. M, Kilian S. G, Potgieter M. A, du Preez, J. C. 2003. The effect of production parameters on the synthesis of the prebiotic trisaccharide, neokestose, by Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Enzyme Microb Technol 32:728–737.



18.Kruger M. C, Brown K. E, Col1eti G, Layton L, Schollum L. M. 2003. The effect of fructooligosaccharides with various degrees of polymerization on calcium bioavailability in the growing rat. Exp Biol Med 228:683-688.

19.Lim J. S, Lee J. H, Kang S. W, Park S. W, Kim S. W. 2007 Studies on production and physical properties of neo-FOS produced by co-immobilized Penicillium citrinum and neo-fructosyltransferase. Eur Food Res Technol 225:457–462.

20.Lim J. S, Park M.C, Lee J. H, Park S. W, Kim S. W. 2005. Optimization of culture medium and conditions for neo-fructooligosaccharides production by Penicillium citrinum. Eur Food Res Techno1221:639-644.

21.Marx S, Koning S, Hartmeier W. 1999. Synthesis of fructooligosaccharides by native, and immobilized levansucrase from Zymomonas mobilis. Thirteenth forum for applied Biotechnology, Gent. Proceedings. Part I, 642: 246–251.

22.Mitsuoka T, Hidaka H, Eida T.1987. Effect of fructo-oligosaccharides on intestinal microf1ora. Die Nahrung 31:427-436.

23.Nakakuki T. 2005. Present Status and Future Prospects of Functional Oligosaccharide Development in Japan. J. Appl. Glycosci. 52:267-271.

24.Nishisawa K, Nakajima M, Nabetani H. 2001. Kinetic study on
transfructosylation by β-fructofuranosidase from Aspergillus niger ATCC 20611 and availability of a membrane reactor for fructooligosaccharide production. Food Sci Technol Res 7:39–44.

25.Ning Y, Wang J, Chen J, Yang N, Jin Z, Xu X. 2010. Production of neo-fructooligosaccharides using free-whole-cell biotransformation by
Xanthophyllomyces dendrorhous. Biores Technol 101:7472-7478.

26.Rao A. V. 1999. Dose-response effects of inulin and oligofructose on intestinal bifidogenesis effects. J. Nutr 129:1442-1445.

27.Rehm J, Willmitzer L, Heyer A. G. 1998. Production of 1-kestose in transgenic yeasts expressing a fructosyltransferase from Aspergillus foetidus. J Bacteriol 180:1305-1310.

28.Roberfroid M. B. 1996. Functional effects of food components and the gastrointestinal system:Chicory fructooligosaccharides. Nutr Rev. 54: 38-42.

29.Roberfroid M. B, Delzenne N. 1998. Dietary fructans. Annu Rev Nutr 18:117-143.



30.Sacha A, Hijum V, Bonthing K, Mare J, Maarel V, Dijkhuzen L. 2001. Purification of novel fructosyltranferase from Lactobacillus reuteri strains 121 and characterization of the levan produced. FEMS Microbiol Lett 205:323–328.

31.Sangeetha P. T, Ramesh M. N, Prapulla S. G. 2004. Production of fructosyl transferase by Aspergillus oryzae CFR 202 in solid-state fermentation using agricultural by-products. Appl Microbiol Biotechnol 65:530–537.

32.Sangeetha P. T, Ramesh M. N, Papulla S. G. 2005. Recent trends in the microbial production, analyses and application of fructooligosaccharides. Trends Food Sci Technol 16:442-457.


33.Sheu D. C, Lio PJ, Chen S. T, Lin C. T, Duan K. J. 2001. Production of
fructooligosaccharides in high yield using a mixed enzyme system of
b-fructofuranosidase and glucose oxidase. Biotechnol Lett, 23:1499–1503.

34.Sheu D. C, Duan KJ, Cheng CY, Bi JL, Chen JY. 2002. Continuous production of high-content fructooligosaccharides by a complex cell system. Biotechnol Prog. 18: 1283–1286.



35.Sheu D. C, Wu C. T, Wang J. Y. 2010. Production of high-content fructooligosaccharides by immobilized Aspergillus japonicus and Pichia
heimii. The 13th Asia Pacific Confederation of Chemical Engineering
Congress, October 5-8, Taipei, Taiwan.

36.Stewart M. L, Timm D. A, Slavin J. L. 2008. Fructooligosaccharides exhibit more rapid fermentation than long-chain inulin in an in vitro fermentation system. Nutrition Res 28:329-334.

37.Stephanus Kilian, Susanna Kritzinger, Catherine Rycroft, Glenn Gibson and James du Preez. 2002. The effects of the novel bifidogenic trisaccharide, neokestose, on the human colonic microbiota. World J Microbiol Biotechnol 18: 637–644.

38.Tatsuya Masukoa, Akio Minami, Norimasa Iwasakib, Tokifumi Majima,
Shin-Ichiro Nishimura, Yuan C. Lee. 2005. Carbohydrate analysis by a phenol–sulfuric acid method in microplate format. Anal. Biochem. 339:69–7

39.Van Loo J, Coussement P, De Leenheer L, Hoebregs H, Smits G. 1995. On the presence of inulin and oligofructose as natural ingredients in the westem diet. Crit Rev Food Sci Nutr. 35:525-552.



40.Van Hijum S. A. F. T, Van Geel-Schutien G. H, Rahaoui H, Van der Maarel M. J. E. C, Dijkhuizen L. 2002. Characterization of a novel fructosyltransferase from Lactobacillus reuteri that synthesizes highmolecular-weight inulin and inulin oligosaccharides. Appl Environ Microbiol. 68:4390-4398.

41.Westhuizen R. J. V. D. 2008. The potentil of neokestose as a prebiotic for broiler chickens. 17.


42.Yasuda H, Shitoh T, Yamano T, Itoh Y, Shimura S. 1986. Identification of neokestose produced from sucrose by an enzyme of Penicillium oxalicum. Agri Biol Chem.50:777-778.

43.Yawei Ning, Jinpeng Wang, Jing Chen, Na Yang, Zhengyu Jin, Xueming Xu . 2010. roduction of neo-fructooligosaccharides using free-whole-cell biotransformation by Xanthophyllomyces dendrorhous. Biores Technol 101:7472–7478

44.Yoshikawa J, Amachi S, Shinoyama H, Fujii T. 2006 .Multiple β-fructofuranosidases by Aureobasidium pullulans DSM2404 and their roles in fructooligosaccharide production. FEMS Microbiol Lett. 265:159-163.


45.Yun J. W. 1996. Fructo-oligosaccharides-occurrence, preparation and application. Enz Micro Technol 9:107-117.

46.王培銘。綜論功能性寡醣。食品工業，第40卷、第6期、第1–3頁。(2008)