酶法合成低聚半乳糖及其在乳粉中的应用
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摘要
本论文对米曲霉中提取的游离β-半乳糖苷酶作用于高浓度的脱盐乳清溶液合成低聚半乳糖的影响因素，合成低聚半乳糖的最佳反应条件，低聚半乳糖的检测方法，低聚半乳糖在奶粉中的应用，低聚半乳糖对双歧杆菌的促进作用和合成低聚半乳糖过程中的动力学模型进行了研究。
首先通过单因素实验确定各因素对低聚半乳糖合成的影响，并通过正交实验，确定了制备低聚半乳糖的最佳反应条件为：温度50℃，反应时间1h，乳糖浓度30％(w/v)，酶浓度0.12％(w/v)，pH 4.5。然后，对低聚半乳糖的分离、检测方法进行了研究。实验证明，低聚半乳糖可以利用快速、简便的薄层层析法得到分离和检测。最佳的分离条件为：薄板为用0.1mol/L磷酸氢二钠溶液制备的硅胶板，活化温度为110℃，时间为1小时，展开剂为：正丁醇：丙酮：水=4∶5∶1。最终，通过高效液相色谱仪准确地测量反应物中低聚糖的含量为23.4％。在合成低聚半乳糖的最佳条件下，利用乳清粉生产低聚半乳糖后，以鲜奶(w)：含有低聚半乳糖的乳清粉(w)=1∶2比例混合得到功能性奶粉，其成分为：蛋白质21.7％、脂肪19.7％、总糖58.6％、低聚半乳糖7.5％。与此同时，论文还对含有低聚半乳糖的乳清粉和含有低聚半乳糖的奶粉对双歧杆菌的作用进行了评价，确定了低聚半乳糖对双歧杆菌起增殖作用的有效加入量，当低聚半乳糖加入量1％以上就有明显的促进作用。通过跟其他奶粉和低聚异麦芽糖比较得到，低聚半乳糖和低聚异麦芽糖对双歧杆菌的促进作用是相同的，而本论文所制备的奶粉对双歧杆菌的促进作用明显高于市售的功能性奶粉和脱脂奶粉。
本论文进一步对采用游离β-半乳糖苷酶合成低聚半乳糖的最佳反应条件下的动力学过程进行了初步的研究。实验数据通过Matlab统计软件进行处理，得到了合成低聚半乳糖酶促反应过程的乳糖消耗和低聚半乳糖形成的动力学反应模型并确定了动力学模型参数。
Preparation of galactooligosaccharides(GOS) by enzymatic synthesis from high concentration of whey and its application on milk powder as functional ingredients were investigated in this paper.
Single factorial experiments were carried out to study the effects of factors on GOS preparation, then optimized the parameters by orthogonal design. The optimized parameters were temperature 50 , reaction time Ih, lactose concentration 30%(w/v), pH4.5, -galactosidase concentration 0.12%. GOS were separated and determined by thin-layer chromatography during the single factor experiments. Thin-layer was made of silica gel with 0.1mol/1 Na2HPO4 and activated at 110 for Ih. The solvent system was butanol:acetone:water=4:5:l. Samples were determined by HPLC during the orthogonal design and GOS content was 23.4% in the end product. The end product was spray dried or mixed with milk and GOS whey powder and GOS milk powder were acquired. The contents of GOS powder milk were protein21.7%, fat!9.7%, saccharide58.6%, and GOS7.5%. In order to evaluate the bifidofactor, functionality of GOS, experiments were carried out in vitro and GOS whey powder and GOS milk powder showed higher bifidobacterium growth activity compared with skimmed milk and commercial functional milk.
Kinetic models of GOS production and lactose hydrolysis were
obtained by analysis of experimental data through Matlab data analysis software. As a result, the experiment data fitted well with these models.
首先通过单因素实验确定各因素对低聚半乳糖合成的影响，并通过正交实验，确定了制备低聚半乳糖的最佳反应条件为：温度50℃，反应时间1h，乳糖浓度30％(w/v)，酶浓度0.12％(w/v)，pH 4.5。然后，对低聚半乳糖的分离、检测方法进行了研究。实验证明，低聚半乳糖可以利用快速、简便的薄层层析法得到分离和检测。最佳的分离条件为：薄板为用0.1mol/L磷酸氢二钠溶液制备的硅胶板，活化温度为110℃，时间为1小时，展开剂为：正丁醇：丙酮：水=4∶5∶1。最终，通过高效液相色谱仪准确地测量反应物中低聚糖的含量为23.4％。在合成低聚半乳糖的最佳条件下，利用乳清粉生产低聚半乳糖后，以鲜奶(w)：含有低聚半乳糖的乳清粉(w)=1∶2比例混合得到功能性奶粉，其成分为：蛋白质21.7％、脂肪19.7％、总糖58.6％、低聚半乳糖7.5％。与此同时，论文还对含有低聚半乳糖的乳清粉和含有低聚半乳糖的奶粉对双歧杆菌的作用进行了评价，确定了低聚半乳糖对双歧杆菌起增殖作用的有效加入量，当低聚半乳糖加入量1％以上就有明显的促进作用。通过跟其他奶粉和低聚异麦芽糖比较得到，低聚半乳糖和低聚异麦芽糖对双歧杆菌的促进作用是相同的，而本论文所制备的奶粉对双歧杆菌的促进作用明显高于市售的功能性奶粉和脱脂奶粉。
本论文进一步对采用游离β-半乳糖苷酶合成低聚半乳糖的最佳反应条件下的动力学过程进行了初步的研究。实验数据通过Matlab统计软件进行处理，得到了合成低聚半乳糖酶促反应过程的乳糖消耗和低聚半乳糖形成的动力学反应模型并确定了动力学模型参数。
Preparation of galactooligosaccharides(GOS) by enzymatic synthesis from high concentration of whey and its application on milk powder as functional ingredients were investigated in this paper.
Single factorial experiments were carried out to study the effects of factors on GOS preparation, then optimized the parameters by orthogonal design. The optimized parameters were temperature 50 , reaction time Ih, lactose concentration 30%(w/v), pH4.5, -galactosidase concentration 0.12%. GOS were separated and determined by thin-layer chromatography during the single factor experiments. Thin-layer was made of silica gel with 0.1mol/1 Na2HPO4 and activated at 110 for Ih. The solvent system was butanol:acetone:water=4:5:l. Samples were determined by HPLC during the orthogonal design and GOS content was 23.4% in the end product. The end product was spray dried or mixed with milk and GOS whey powder and GOS milk powder were acquired. The contents of GOS powder milk were protein21.7%, fat!9.7%, saccharide58.6%, and GOS7.5%. In order to evaluate the bifidofactor, functionality of GOS, experiments were carried out in vitro and GOS whey powder and GOS milk powder showed higher bifidobacterium growth activity compared with skimmed milk and commercial functional milk.
Kinetic models of GOS production and lactose hydrolysis were
obtained by analysis of experimental data through Matlab data analysis software. As a result, the experiment data fitted well with these models.
引文
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[11] Pazur J H. The enzymatic conversion of lactose into oligosaccharides[J]. Science, 1953,117:355～368.
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[2] Zarate S, Lopez-Leiva M H.Oligosaccharide formation during enzymatic lactose hydrolysis:a literature review[J]. J Food Protection 1990, 53(3):262～268.
[3] Yanahira S,Kobayashi T, Suguri T et al, Structures of novel acidic galactooligosaccharides synthesized by Bacillus circulans beta-galactosidase[J]. Biosci Biotech Biochem, 1998, 62 (9) 1791-1794
[4] Toba T, Yokota A. Adavchi S. Oligosaccharide structures formed during the hydrolysis of lactose by Aspergillus oryzae β-galctosidase[J]. Food Chem, 1985,16:147～162.
[5] Mozaffar Z, Nakanishi K, Matsuno R. Formation of oligosaccharides during hydrolysis of lactose in milk using beta-galactosidase from Bacillus circulans[J]. Journal of food science. 1984,48:3053～3061.
[6] Ozawa,O.,ohtsuka,K.,& Uchida, R. Production of 4'-galactosyllactose by mixed cells of Cryptococcus laurentii and baker's yeast[J]. Nippon Shokuhin Kogyo Gakkaishi. 1989,36:898～902
[7] 郑建仙，李璇．功能性低聚糖[J]．中国食品工业，1998，5(12)：26～28
[8] Voragen, A. G. J. Technological aspects of functional food-related carbohydrates[J]. Trends in Food science and Technology, 1998.9:325～335
[9] 陈瑞娟．新型低聚半乳糖的介绍[J]．食品与发酵工业，1993(3)：82～90
[10] Aronson M. Transgalactosidation during lactose hydrolysis[J]. Archives of Biochemistry and Biophysics, 1952,39: 370～378.
[11] Pazur J H. The enzymatic conversion of lactose into oligosaccharides[J]. Science, 1953,117:355～368.
[12] 郑建仙．功能性食品(第一卷)[M]．中国轻工业出版社，1999．8
[13] Bouhnik, Y., B. etc. Administration of transgalactooligosaccharides increases in fecal bifidobacteria and modifies colonic fermentation metabolism in healthy humans[J].Journal of Nutrition. 1997,127:444～448
[14] 马廷和，周培瑾．浅谈寡糖的开发和应用[J]．食品与发酵工业，1992，1：80～82．
[15] 郑建仙，耿立萍．功能性低聚糖析论[J]．食品与发酵工业，1997，23(1)：39～46．
[16] M., deguchi, Y., Matsumoto, K., Kimura, M., Onodera, N.,& Yajima, T. Influence of galactooligosaccharides on the human fecal microflora[J]. Journal of nutritional science and vitaminology, 1993,39:635～640.
[17] Ohtuka, K., Tsuji, K etc. Availability of 4′-galactosyllactose in rat[J].Journal of Nutritional Science and Vitaminology. 1990,36:265～276
[18] Chonan O, Matsumoto K, Watanuki M. Effect of galactooligosaccharides on calcium absorption and preventing bone loss in ovariectomized rats [J]. Biotech. Biochem., 1995,59(2):236～239.
[19] Chonan O, Watanuki M. Effect of galactooligosaccharides on calcium absorption in rats[J]. J.Nutr. Sci. Vitaminol., 1995, 41:95～104
[20] 归莉琼，魏东芝．生物活性物质—低聚半乳糖[J]．中国乳品工业，1999，31 (2)：171～174．
[21] 归莉琼，魏东芝，俞俊棠．米曲霉半乳糖苷酶催化合成低聚半乳糖[J]．华东理工大学学报，1998，24(4)：422～426．
[22] 尤新．功能性食品配料—新型低聚半乳糖[J]．食品科学，1995，(11)：41-44．
[23] 尤新．功能性发酵制品[M]．中国轻工业出版社，2000．
[24] Vassilis G,Migues L. Extraction of beta-galactodase from different microorganisms[J]. Process Biochemistry, 1985,12:2～12
[25] Ajisaka K, Nishida H, Fujimoto H. The synthesis of oligosaccharides by the reversed hydrolysis reaction of beta-glucosidase at high substrate concentration and at high temperature[J]. Biotechno lett. 1987,9:243～248.
[26] Nakanishi K, Matsuno R, Torii K, Yamamoto K, Kamikubo T. Properties of immobilized beta-D-galactosidase from Bacillus circulans[J]. Enzyme microb technology, 1983,5:115～120.
[27] Norimasa O, Takshi T. Purification and properties of a novel thermostable galactooligosaccharide-producing β-galactosidase from sterigmatomyces elviae CBS8119[J].Applied Enviro Microbio. 1995,61 (1): 4025～4030
[28] Lind D L, Daniel. R M Cowan D A, et al. β-galactosidase from a strain of the anaerobic thermophile, thermoanaerobacter[J].Enzyme Microb Technol, 1989,11:180～188
[29] Mahoney R R. In Advanced Dairy Chemistry[M]. London: Chapman and Hall, 1996, 3:77～126.
[30] Prenosil J E. Stuker E, Bourne J R. Formation of oligosaccharides during enzymatic lactose hydrolysis Ⅰ. State of art[J].Biotechnol Bioeng, 1987,30:1029～1025.
[31] Gekas V, Lopez-leiva, M. Hydrolysis of lactose:A literature review[J]. Process Biochem, 1995,2:2～12
[32] Roberts H R,Pettinati J P. Concentration effects in the enzymatic conversion of lactose to oligosaccharides[J]. Agri Food CHEM, 1957,5(2): 130～134.
[33] Kan T, Kobayashi Y. United states Patent[p],No: 4957860.
[34] Mozaffar Z, et al. Appl. Continuous production of galacto-oligosaccharides from lactase using immobilized β-galactosidase from Bacillus circulans [J]. Microbiol Biotechnol. 1986,25:224～228.
[35] Shin H-J, Yang J W. GOS production by β-galactosidase in hydrohpobie organie media[J]. Biotechnol Lett, 1994,17(10):1157～1162.
[36] Onish N, Yamashiro, A and Yokozeki, K. Production of galacto-oligosaccharide from lactose by Sterigmatomyces elviae[J]. Applied and Environmental Microbiology, 1995,61:4022～4025
[37] Manley H, Geoffery N, et al. Mechanism for the production of oligosaccharides by thermalization[J]. Carbohydrate Research, 1993,24:183
[38] 张唯杰．复合多糖的生化研究技术[M]．上海科技出版社，1987．
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