摘要
本课题以米糠为原料，通过酶法水解制备米糠分离蛋白，研究了提取米糠蛋白所需的酶类，以及最佳水解工艺，并对米糠蛋白的功能性质作初步的探讨。
试验所采用的原料为黑龙江五常稻米加工厂的米糠，对原料进行了测定，结果为蛋白质：12.28％、脂肪：20.01％、纤维素：23.95％、淀粉：15.44％、水分：10.78％。
酶本身就是一种蛋白质，因此在提取米糠蛋白的过程中，要选择合适的方法灭酶，通过试验，选择灭酶方式为：调pH=4，50℃，30min。
关于米糠蛋白的等电点，各文献中没有详细报道，本试验采用测定不同pH下米糠蛋白浓度的方法，对米糠蛋白的等电点进行了测定，结果米糠蛋白的等电点为pH=4.6。
不同的酶水解米糠有不同的作用方式，对细胞破壁酶类和蛋白酶类进行选择，以蛋白收率为指标，水解度为参考指标，比较各种酶水解米糠制蛋白的效果，认为纤维素酶和复合蛋白酶水解效率高，因此选择这两种酶为水解用酶。
分别对纤维素酶、复合蛋白酶、植酸酶的水解效果进行分析，在pH、温度为各酶最适条件，时间、底物浓度相同的情况下，采用酶添加量的单因素试验，比较在最佳酶用量的情况下的蛋白收率，结果蛋白收率不是很高。同时根据各种酶最佳用量，可确定配料试验中各种酶的添加范围。
采用配料试验，将纤维素酶、复合蛋白酶、植酸酶的添加量范围折合成每种酶所占的百分比，应用二因素二次旋转正交回归设计，在底物浓度15％、温度45℃、pH=5.5、添加量2％、时间2小时的条件下，以蛋白收率为指标，水解度为参考指标，建立回归模型y=70.76+1.58X_1+2.40X_2-0.9X_1~2+1.28X_1X_2
y：蛋白收率 X_1：纤维素酶添加量 X_2：复合蛋白酶添加量
通过检验证实回归模型与实际情况拟和较好，通过频数分析及变量变换，选择的各种酶用量的百分比分别为纤维素酶为65.84％、蛋白酶为18.52％、植酸酶为15.64％。
采用正交试验和单因素试验，确定复合酶的反应条件为，温度：45℃、时间2小时、底物浓度20％、酶添加量4％、pH=5.5。
测定米糠蛋白的乳化性、起泡性、粘度，并与大豆蛋白进行了比较，结果米糠蛋白的乳化性优于大豆蛋白，而起泡性和粘度不及大豆蛋白。
The research was conducted on enzyme hydrolysis in preparing protein from rice bran. Several enzymes were selected and hydrolysis technique was optimized. The functional properties of rice bran protein were primary studied.
The material was produced by Wu Chang Rice Manufactory. The composition of rice bran was that protein: 12.28%, fat:20.01%% cellulose: 23.95%, starch: 15.44%, moisture: 10.78%.
Enzyme is a kind of protein, so the suitable method was selected by testing, which can effectively inactivate enzyme, it was that pH:4.0, temperature:50 C, time:3h.
As pI of rice bran protein was not reported by references. It was determined by testing protein concentration at different pH. The result of rice bran protein pI was pH=4.6.
Different enzyme has different hydrolysis process. The kind of broken kernels enzymes and proteases were selected in the process of preparing rice bran protein. Comparing nitrogen reclaim and degree of hydrolysis, cellulase and protamex were fitted.
Cellulase , protamex, phytase were differently used to hydrolysis rice bran under the optimum temperature and pH, the time and substrate concentration were under the same condition. Single factor was adopted to determine optimum enzyme concentration of every enzyme, and the result showed nitrogen reclaim of single enzyme was not high under the optimum condition. At the same time, in the ingredient test the range of every enzyme concentration was determined by above result.
In the ingredient test, the range of single enzyme concentration was transformed to the percent of total. Then revolving, orthogonal and tropical design of the second power of two factors was adopted to study the percent of cellulase , protamex and phytase. Under the condition of substrate concentration 15%, temperature 45 C,pH=5.5,time 2h and enzyme concentration 2%, the model was established: y =70.76+1.58X1+2.40X2-0.9X12+1.28X1X2
y: nitrogen reclaim X1: cellulase concentration X2: protamex concentration
F-test was used to verity the model. The result indicated the testing goodness of it. The result of frequency analysis and variable transform showed that the optimum enzyme proportion was that cellulase 65.84%; protamex 18.52%; phytase 15.64%.
The orthogonal experiment and single factor test were used to determined the optimum condition of multiple enzyme, it was substrate concentration20%, temperature 45"C,pH=5.5,time 2h, enzyme concentration 4%.
Emulsion capacity, foaming capacity and viscosity of rice bran protein were tested and
were compared to soybean protein. The results were that emulsion capacity of rice bran protein was higher than soybean protein, and foaming capacity and viscosity were lower than it.
Postgraduate: Wang Xuefei Major: Food Science Supervisor: Associate Prof Yu Guoping
引文
1 毕士峰，张毅．一种新的食品添加剂——植酸酶．食品科学．2000，21(8)：9～10
2 蔡东联．对米糠营养价值的再认识．中国食物与营养．2000(2)：31
3 陈季旺，姚惠源，张小勇等．米糠可溶性蛋白的提取工艺和特性研究．中国油脂．2003，28(2)：46～50
4 陈正行，姚惠源，周素梅．米蛋白和米糠蛋白开发利用．粮食与油脂．2002(4)：6～9
5 陈正行，姚惠源．米糠健康食品的研究．无锡轻工大学学报．2001，20(2)：202～204
6 陈正行，周彤．米糠：一种潜在的健康食品优质原料．粮食与饲料工业．1999(10)：40～45
7 陈正行．米糠浓缩蛋白提取及功能性评价．无锡轻工大学学报．食品与发酵工业．2000，26(4)：17～19
8 陈正行．米糠脂多糖提取物中分离蛋白质的研究．中国粮油学报．2000，15(5)：7～9
9 高红岩，石彦国，马永强．纤维素酶对高变性大豆蛋白溶出率影响的研究．食品科技．2001(1)：22～24
10 龚院生，姚惠源，杭敏．米糠水溶性提取物γ-谷维醇的富集合米糠保健饮料工艺研究．食品科学．2001，22(3)：47～49
11 顾华孝．米糠的食用性和在保健功能食品上的应用．粮食与饲料工业．2001(5)：46～48
12 韩丽华．碱溶酸沉法生产大豆分离蛋白及影响质量、出率的主要因素．中国油脂．1998，23(6)：29～31
13 胡国华，余迎利，黄绍华．米糠半纤维素的研究及应用．粮食与饲料工业．1998(2)：42～43
14 胡国华，翟瑞文．脱脂米糠半纤维素A分离与鉴定．粮食与油脂200(4)：10～11
15 华欲飞，顾玉兴．大豆蛋白的吸水性和持水性．中国油脂．1999，24(4)：64～66
16 李汴生，彭志英，宁正祥．植物油料取油的酶预处理工艺．中国粮油学报．1997，12(6)：24～30
17 李正明，王兰军．植物蛋白生产工艺与配方．中国轻工业出版社，1998：33～41
18 刘军海，刘斌，刘敏等。米糠浸出和米糠油精炼技术研究进展。中国油脂。2001，26(3)：17～20
19 刘玮，张劲松，王兴礼等．米糠的深加工及综合利用．现代化农业．2000(11)：19～20
20 刘雄，阚健全，陈宗道．米糠蛋白的功能特性和应用．粮食与饲料工业．2001(12)：35～37
21 刘志强，何昭青．水酶法花生蛋白提取及制油研究．中国粮油学报．1999，14(1)：36～39
22 裘爱泳．酶法水解米糠纤维以制备保健食品的研究．粮食与饲料工业．1997(11)：37～39
23 宋金彩，单安山．植酸酶对蛋白质和氨基酸利用率的影响．中国饲料．2000(10)：14～15
24 唐传核．米糠油来源生育三烯酚生理功能作用．粮食与油脂．2000(2)：35～36
25 王洪新等．食品新资源．中国轻工业出版社，2002：1～5
26 王立，陈正行，王文高．米糠蛋白研究．粮食与油脂．2002(2)：28～29
27 王立，陈正行，姚惠源．酶法提取米糠蛋白质的研究．粮食与油脂．2002(4)：12～13
28 王立，周素梅，陈正行．提取米糠蛋白的探讨．西部粮油科技．2002(3)：35～37
29 王文高，陈正行，姚惠源．不同蛋白酶提取大米蛋白质的研究．粮食与饲料工业．2002(2)：41～42
30 王文高，陈正行，姚惠源．大米蛋白及其水解物功能性质与疏水性关系的研究．粮食与饲料工业．2002，27(7)：49～50
31 王亚林，陶兴无，钟放旭等。碱酶两步法提取米渣中蛋白质的工艺研究．中国油脂．2002，27(3)：53～54
32 王璋，许时婴，林岚等．酶法从全酯大豆中同时制备大豆油和大豆水解蛋白工艺的研究．无锡轻工大学学报．1994，13(3)：179～191
33 魏述众，关凤梅，韩应昌等．“大豆分离蛋白”工艺用复合酶制剂的研究．工业微生物．1999，29(1)：11～15
34 吴莺，姚惠源．米糠半纤维素降胆固醇机理的研究．粮食与油脂．2001(1)：33～35
35 谢良，王璋，蔡宝玉．大豆分离蛋白的组成与功能性质．中国粮油学报．2000，15(6)：6～10
36 邢小鹏，吴高峻，孙华．大豆分离蛋白的功能性质．食品工业科技．2000，21(4)：74～76
37 徐红华，于国平．米糠蛋白的分离及其营养特性的研究．粮油食品科技．2002，10(1)：17～18
38 严岩，张全福．纤维素酶的性质、应用及其环保意义．农业环境与发展．1997(1)：17～20
39 颜季琼．高等植物细胞壁的结构和功能．生物学通报．1999，37(1)：6～7
40 姚惠源，周素梅，王立．米糠与米糠蛋白质的开发与利用．无锡轻工大学学报．2002，21(3)：312～316
41 姚惠源．米糠脱酶稳定化技术和米糠食品的开发研究．粮食与油脂．2002(4)：2～5
42 余蜀宜，王传槐．纤维素酶降解大豆细胞壁生产豆腐的研究．食品科学．2002，23(4)：79～82
43 张晖，毛锦生，张建萍．小麦麸皮蛋白质的提取与应用．无锡轻工大学学报1998，17(1)：44～48
44 赵新淮，冯志彪．蛋白质水解物世界度的测定．食品科学．1994(11)：65～67
45 周雅萍，姚惠源，陈正行．米糠营养食品的研究开发．无锡轻工大学学报．1999，18(3)：52～56
46 朱文华，姚惠源．全脂米糠的积压稳定性．无锡轻工大学学报．2000，19(6)：557～559
47 A. Proctor, D. J. Bowen. Ambient-Temperature Extraction of Rice Bran Oil with
Hexane and Isopropanal. Journal of American Oil Chemistry Society. 1996, 73(6):811～813
48 Ansharullah, James A Hourigan, Colin F Chesterman. Application of Carbohydrases in Extracting Protein from Rice Bran. Journal of Science Food Agriculture. 1997, 74, 141～146
49 Bocevska M, Dj. Karlovic, J Turkulov. Quality of Corn Germ Oil Obtained by Aqueous Enzymatic Extraction. Journal of American Oil Chemistry Society. 1993, 70(6): 1273～1277
50 Cagampang. G. B, Cruz. L.J. Study on the Extraction and Composition of Rice Proteins. Cereal Chemistry. 1996, 43(2): 145～147
51 Chen W P, Chang Y C. Production of High Fructose Rice Syrup and High Protein Rice Flour from Broken Rice. Journal of Science Food Agriculture. 1984, 35(8): 1128～1135
52 Don. R Mccaskill, Fan Zhang. Use of Rice Bran Oil in Foods. Food Technology. 1999, 53(2): 50～53
53 Frederick F Shih, Kim Daigle. Use of Enzyme for the Separation of Protein from Rice Flour. Journal of American Oil Chemistry Society. 2000, 77(8): 239～245
54 Frederick F Shih. Use of Enzymes for the Separation of Protein from Rice Four. Cereal Chemistry. 1997, 74(4): 437～441
55 Frederick F. Shih, Kim W. Daigl. Preparation and Characterization of Rice Protein Isolates. Journal of American Oil Chemistry Society. 2000, 77(8):885～889
56 J. S. Hamada. Characterization and Functional Properties of Rice Bran Proteins Modified by Commercial Exoproteases and Endoproteases. Journal of Food Science. 2000, 65(2): 305～310
57 J. S. Hamada. Characterization of Protein Fractions of Rice Bran to Devise Methods of Protein Solubilization. Cereal Chemistry. 1997, 74(5):662～668
58 M. Wang, Hettiarachchy N S. Preparation and Functional Properties of Rice Bran Protein Isolate. Journal of Science Food Agriculture. 1999, 47, 411～416
59 Marita T, Kiriyama S. Mass Production Method for Rice Protein Isolation and Nutritional Evaluation. Journal of Food Science. 1993, 56(4): 1393～1396
60 Proctor. A, V. M. Jackso, M. Scott, etc. Rapid Equilibrium Extraction of Rice Bran at Ambient Temperature. Journal of American Oil Chemistry Society. Soc. 1994, 71: 1295～1296
61 R. Sengupta. Enzymatic of Mustard Seed and Rice Bran. Journal of American Oil Chemistry Society. 1996, 73(6):687～692
62 Ramsay. M. E. J. T Hsu, R. A. Novak. Processing of Rice Bran by Supercritical Fluid Extraction. Food Technology. 1991, 45(2):98～104
63 Ravin Gnanasambandam. Protein Concentrate from Unstabilized and Stabilized Rice Bran: reparation and Properties. Journal of Food Science. 1995, 60(5):1066～1069
64 Roberts P J, Simmond D H. Extraction of Protein and Solid from Wheat Bran. Journal of Science Food Agriculture. 1985, 36(1): 5～10
65 Tatsuya Morita, Shuhachi Kiriyama. Mass Production Method for Rice Protein Isolate and Nutritional Evaluation. Journal of Food Science. 1993, 58(6): 1393～1396