玉米蛋白酶解制备高F值低聚肽及应用研究
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摘要
高F值低聚肽是蛋白酶作用于食物蛋白后形成的一种低分子量生理活性肽。因其具有独特的氨基酸组成和生理功能而受到食品和医药界的高度关注。本研究主要是以玉米淀粉副产物——玉米蛋白粉为原料，通过酶法水解，制备高F值低聚肽，深层次开发玉米蛋白，提高玉米资源的利用率。
首先对水解玉米蛋白的酶进行了筛选工作，以水解度及游离氨基酸含量为指标，选出Alcalase为较佳的蛋白酶。
采用了四因素三水平L_9(3~4)正交试验设计确定Alcalase蛋白酶水解玉米蛋白的最佳条件为：pH值8.0，温度55℃，酶与底物浓度比3％，底物浓度5％；对水解时间进行单因素分析，确定最佳水解时间为4h，此时水解度可达11.62％。
对水解液超滤工艺参数的研究结果为：选用NMWL10000膜效果较好，超滤的适宜压力为20KPa。
水解物含盐量较高，用阴阳离子交换树脂脱盐时，流速为8倍柱体积/小时的效果较好，氮回收率为81.67％，脱盐率达80.95％。
活性炭吸附色谱法去除游离芳香族氨基酸的条件为：7号活性炭，活性炭与物料比1：12，温度25～35℃，pH值为2.0～3.0，静态吸附；活性炭洗脱条件为：2mol/L氨水和无水乙醇以等体积混合，动态洗脱。
对吸附后的低聚肽溶液进行分析，紫外扫描结果表明活性炭对芳香族氨基酸吸附效果好；根据氨基酸分析结果得到的低聚肽F值为34.71，游离氨基酸含量6.42％，产品得率为11.59％。产品为澄清透明的溶液，无苦味和异味，含盐量较少。
采用G—25凝胶过滤层析法测定高F值低聚肽分子量，结果表明：高F值低聚肽的肽分子分布是连续的，分子量大多集中在1000～1300之间。
以制得的高F值低聚肽溶液为基料，研制肽饮料和酸奶，肽饮料最佳配方为高F值低聚肽溶液1.2％，果汁4％，砂糖6％，柠檬酸0.15％。最佳营养酸奶配方为低聚肽溶液20％，接菌量3％，砂糖6％。产品感观质量好，理化指标和微生物指标合格。
High fischer ratio oligo-peptide is a kind of low molecular weight physiological active peptide. It is the production that protease processed the food protein. Because the High fischer ratio oligo-peptide has specially amino acids composition and physiological activity, food and curative domain pay more attention to it. This thesis was conducted on preparation of high fischer ratio oligo-peptide by protease hydrolysis from Corn gluten meal, a byproduct of the wet-milling process, to improve the utilization of corn resource.
First, the performance of enzymes was compared. With the index of DH and content of free amino acids, Acalase was selected as the suitable protease to prepare hydrolysate.
Orthogonal design (L9(3)4) was adopted to optimize the conditions of hydrolysis. The result was prefered under the condition of pH 8.0, temperature 55 C, ratio of enzyme to substrate 3% and substrate concentration 5%. Simple factor analysis indicated that the optimal time of hydrolysis was 4 hours and the hydrolysate with 11.62% DH was obtained.
Based on the comparison of operation parameters, NMWL10000 membrane was used for ultrafiltration of corn gluten hydrolysate and ulrrafiltration pressure was 20KPa.
Ion exchange resin was used to remove salt in the hydrolysate. The performance of the treatment was better when flow rate of the hydrolysate was eight times of column volume per hour. The nitrogen retention of the hydrolysate was 81.67% and the ratio of desalinization was 80.95% after passing the ion exchange resin.
No.7 active carbon was used to remove free aromatic amino acids by the method of stable absorption. The engineering conditions have been investigated. The optimal conditions were proposed that the ratio of the weight of active carbon to volume of hydrolysate was 1:12, the temperature was 25-35 C and the value of pH was 2.0-3.0. Dynamic elution solution contained 50% 2mol/L ammonia water and 50% ethanol.
The result of UV-scanning indicated that active carbon was efficient to absorb aromatic amino acids. The consequence of amino acid analysis showed that the F value of oligo-peptides was 34.71, the content of free amino acid was 6.42%, ultima recovery of product was 11.59%. The product appeared a transparent solution without bitter taste and undesirable flavor, and the content of salt was little.
G-25 gel filtration chromatography was used to determination the molecular weight of the high F value oligo-peptides. The result showed that the distribution of molecular weight of the high F value oligo-peptides was consecutive. It ranged from 1000 to 1300 .
The studies of peptide beverage and yoghurt were based on high F value oligo-peptides. The formula of nutritive beverage was composed of 1.2% high F value oligo-peptide solution, 4% fruit juice, 6% sugar and 0.15% citric acid. The optimum formula of yoghurt was 20% high
F value oligo-peptide solution, 3% culture starter, 6% sugar. The sensory quality of products was well and physical and chemical index were up to standard.
Postgraduate: LinLi Major: Food Science Supervisor: Prof. MaYing
首先对水解玉米蛋白的酶进行了筛选工作，以水解度及游离氨基酸含量为指标，选出Alcalase为较佳的蛋白酶。
采用了四因素三水平L_9(3~4)正交试验设计确定Alcalase蛋白酶水解玉米蛋白的最佳条件为：pH值8.0，温度55℃，酶与底物浓度比3％，底物浓度5％；对水解时间进行单因素分析，确定最佳水解时间为4h，此时水解度可达11.62％。
对水解液超滤工艺参数的研究结果为：选用NMWL10000膜效果较好，超滤的适宜压力为20KPa。
水解物含盐量较高，用阴阳离子交换树脂脱盐时，流速为8倍柱体积/小时的效果较好，氮回收率为81.67％，脱盐率达80.95％。
活性炭吸附色谱法去除游离芳香族氨基酸的条件为：7号活性炭，活性炭与物料比1：12，温度25～35℃，pH值为2.0～3.0，静态吸附；活性炭洗脱条件为：2mol/L氨水和无水乙醇以等体积混合，动态洗脱。
对吸附后的低聚肽溶液进行分析，紫外扫描结果表明活性炭对芳香族氨基酸吸附效果好；根据氨基酸分析结果得到的低聚肽F值为34.71，游离氨基酸含量6.42％，产品得率为11.59％。产品为澄清透明的溶液，无苦味和异味，含盐量较少。
采用G—25凝胶过滤层析法测定高F值低聚肽分子量，结果表明：高F值低聚肽的肽分子分布是连续的，分子量大多集中在1000～1300之间。
以制得的高F值低聚肽溶液为基料，研制肽饮料和酸奶，肽饮料最佳配方为高F值低聚肽溶液1.2％，果汁4％，砂糖6％，柠檬酸0.15％。最佳营养酸奶配方为低聚肽溶液20％，接菌量3％，砂糖6％。产品感观质量好，理化指标和微生物指标合格。
High fischer ratio oligo-peptide is a kind of low molecular weight physiological active peptide. It is the production that protease processed the food protein. Because the High fischer ratio oligo-peptide has specially amino acids composition and physiological activity, food and curative domain pay more attention to it. This thesis was conducted on preparation of high fischer ratio oligo-peptide by protease hydrolysis from Corn gluten meal, a byproduct of the wet-milling process, to improve the utilization of corn resource.
First, the performance of enzymes was compared. With the index of DH and content of free amino acids, Acalase was selected as the suitable protease to prepare hydrolysate.
Orthogonal design (L9(3)4) was adopted to optimize the conditions of hydrolysis. The result was prefered under the condition of pH 8.0, temperature 55 C, ratio of enzyme to substrate 3% and substrate concentration 5%. Simple factor analysis indicated that the optimal time of hydrolysis was 4 hours and the hydrolysate with 11.62% DH was obtained.
Based on the comparison of operation parameters, NMWL10000 membrane was used for ultrafiltration of corn gluten hydrolysate and ulrrafiltration pressure was 20KPa.
Ion exchange resin was used to remove salt in the hydrolysate. The performance of the treatment was better when flow rate of the hydrolysate was eight times of column volume per hour. The nitrogen retention of the hydrolysate was 81.67% and the ratio of desalinization was 80.95% after passing the ion exchange resin.
No.7 active carbon was used to remove free aromatic amino acids by the method of stable absorption. The engineering conditions have been investigated. The optimal conditions were proposed that the ratio of the weight of active carbon to volume of hydrolysate was 1:12, the temperature was 25-35 C and the value of pH was 2.0-3.0. Dynamic elution solution contained 50% 2mol/L ammonia water and 50% ethanol.
The result of UV-scanning indicated that active carbon was efficient to absorb aromatic amino acids. The consequence of amino acid analysis showed that the F value of oligo-peptides was 34.71, the content of free amino acid was 6.42%, ultima recovery of product was 11.59%. The product appeared a transparent solution without bitter taste and undesirable flavor, and the content of salt was little.
G-25 gel filtration chromatography was used to determination the molecular weight of the high F value oligo-peptides. The result showed that the distribution of molecular weight of the high F value oligo-peptides was consecutive. It ranged from 1000 to 1300 .
The studies of peptide beverage and yoghurt were based on high F value oligo-peptides. The formula of nutritive beverage was composed of 1.2% high F value oligo-peptide solution, 4% fruit juice, 6% sugar and 0.15% citric acid. The optimum formula of yoghurt was 20% high
F value oligo-peptide solution, 3% culture starter, 6% sugar. The sensory quality of products was well and physical and chemical index were up to standard.
Postgraduate: LinLi Major: Food Science Supervisor: Prof. MaYing
引文
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2 陈石良，吴建平等．高F值寡肽的研究进展．食品与机械．1998，2：12～14
3 崔凌飞，王遂．蛋白质及其水解物的分析应用．哈尔滨商业大学学报．2002，18(1)：117～120
4 冯志彪，张伟．螺旋藻蛋白质水解液的精制．食品工业．2002，3：3～4
5 谷文英．肝性脑病防治肽——高F值低聚肽的研究．中国食品添加剂．2000，2：69～73
6 郭俊生，赵法汲等．大豆高支低芳氨基酸混合物在肝功不全治疗中的应用．营养学报．1990，12(2)：127～131
7 郭兴凤．蛋白质水解度的测定．中国油脂．2000，(25)6：176～177
8 胡学烟．玉米蛋白粉开发与利用．粮食与油脂．2001，11：32～33
9 纪庆芳．活性炭色谱法分离苯丙氨酸、酪氨酸．氨基酸杂志．1989，3：1～3
10 蒋关昌．发酵调味品生产技术．中国轻工业出版社，1999：70～76
11 蒋滢．肝性脑病与氨基酸．氨基酸杂志．1990，1：35～36
12 李和平，李梦琴等．新型食品添加剂玉米蛋白发泡粉的研制．郑州粮食学院学报．1998，3：33～37
13 李琰．玉米蛋白发泡粉生产新工艺的研究．粮食与饲料工业．2002，3：40～42
14 马莺．大豆低聚糖的提取及酶改性的研究．东北农业大学博士论文．2000，6：23～24
15 马永强，赵毅．大豆蛋白水解物中肽分子分布的研究．中国粮油学报．2001，(16)3：15～17
16 牟德华，李艳等．玉米综合加工技术．食品科学．2002，23(8)：357～362
17 任国谱，谷文英．控制酶解黄粉蛋白制备富含“条件必需氨基酸’—谷氨酰胺活性肽营养液的研究．中国粮油学报．200116(2)：9～12
18 邵继智．支链氨基酸的生化与营养．氨基酸杂志．1990，2：23～35
19 沈蓓英，孙冀平．玉米蛋白深层次开发和高F值肽．2000：131～137(会议论文)
20 沈蓓英．高F值寡肽生理功能和制备．粮食与油脂．1999，2：27～30
21 沈蓓英，玉米蛋白深层次开发．粮食与油脂．1998，3：39～40
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