摘要
本文以低温冷榨油茶籽粕为原料,对油茶籽粕蛋白提取工艺及其功能性质进行了研究。利用优化工艺提取的油茶籽粕蛋白,酶解制备油茶籽多肽及血管紧张素转化酶(angiotensin converting enzyme, ACE)抑制肽,优化了制备多肽及ACE抑制肽的工艺,利用高效液相色谱对多肽的分子量分布及氨基酸组成进行了测定和分析,通过体外模拟试验研究了油茶籽多肽对活性氧自由基清除的效果。主要研究结论如下：
通过L9(34)正交实验,确定最佳提取参数为：料液比为1：25,pH值为10,浸提时间为80min,浸提温度为45℃,蛋白提取率达57.8%。
以水解度为指标,采用四因子二次回归正交旋转组合试验,对中性蛋白酶酶解油茶籽粕蛋白的研究建立起的相关回归模型为：Y=-205.520625-0.000581*X1+2.240417*X2+46.334167*X3+0.261667*X4-0.000000177*X12+0.000012692*X1*X2-0.026592*X22+0.000233*X1*X3+0.004500*X2*X3-3.434167*X32+0.000265*X1*X4+0.027000*X2*X4+0.275000*X3*X4-0.649167*X42
中性蛋白酶水解油茶籽蛋白的最佳反应条件是：酶解温度46.8℃,酶解时间4.3h,酶解pH7.2,底物浓度3.0%,加酶量8028.2u／g底物,水解度达到12.4%。
油茶籽粕蛋白的氨基酸总量为43.38g／100g蛋白质,其中谷氨酸含量最高为9.67g／100g蛋白质,油茶籽粕蛋白大部分不溶于水,而溶于稀碱液。蛋白质的等电点在pH5左右。
油茶籽多肽的分子量主要集中在3000Da以下,其中分子量1000Da以下所占面积最大,达到83.73%,肽链的平均长度PCL为8.4,平均相对分子质量Mw为942.3Da。超滤(Mw3000Da)后,分子量<1000Da的小分子肽所占的比率大幅度提高,上升到97.54%。肽链的平均长度PCL为8.06,平均相对分子质量Mw为905.1Da。
采用三因素二次回归正交旋转组合试验,建立ACE抑制肽的抑制率与温度、底物浓度、酶与底物的质量比三因子的数学回归模型：Y=-700.499176+30.442000*X1+5.646534*X2-2.555909*X3-0.323605*X12+0.002000*X1*X2-0.698722*X22+0.236841*X1*X3+0.112727*X2*X3-1.124574*X32
ACE抑制肽制备的最佳反应条件是：酶解温度48.6℃,底物浓度4.4%,酶与底物的质量比4.2%,酶解pH7.2,酶解时间3h,此时ACE抑制理论最大值为46.24%。
通过采用脱氧核糖法、邻苯三酚白氧化法、分光光度法分别考察了油茶籽多肽清除羟基自由基、DPPH自由基、超氧阴离子自由基的能力,结果显示油茶籽多肽对·OH.DPPH.、O2-等自由基均有清除作用,且油茶籽多肽浓度增大清除能力增大。
In this paper, the extraction technology and the functional properties of cold pressing camellia seeds-cake protein were studied. The preparation techniques & the optimal extraction process of camellia seeds-cake peptides and ACE inhibitory peptide through hydrolyzing camellia seeds-cake protein by neutrase were studied, molecular weight distribution and amino acids composition of polypeptides were determined and analyzed by high performance liquid chromatography. The simulation experiments in vitro were used to determine the effects of camellia seeds-cake peptides on scavenging active oxygen free radicals. The main findings are as follows:
Through orthogonal and optimization experiments, the optimal conditions for protein extraction were:The leaching solution pH value,10.0; extraction temperature, 45℃; grinding degree of teacake,80 mesh; feed-liquid ratio,1 :25; leaching,80 min and pH value of extracting solution,4.5.At this condition, protein extraction rate was 57.8%.
Through four-factor quadratic regression orthogonal rotation experiments, a mathmatical model was set up as follow:
Y=-205.520625-0.000581*X1+2.240417*X2+46.334167*X3+0.261667*X4-0.00 0000177*X1*X1+0.000012692*X1*X2-0.026592*X2*X2+0.000233*X1*X3+0.004500 *X2*X3-3.434167*X3*X3+0.000265*X1*X4+0.027000*X2*X4+0.275000*X3*X4-0.6 49167*X4*X4
Based on this model, the optimal conditions for neutrase enzymolysis were: enzymolysis temperature 46.8℃, enzymolysis time 4.3 hours, enzymolysis pH value 7.2, substrate concentration 3.0%,and the amout of enzyme 8028.2 u/g substrate, the degree of hydrolysis was up to 12.4%.
The total amino acids of camellia seeds-cake protein was 43.38 g/100g protein, and the glutamic acid was up to 9.67 g/100g protein. To the camellia seeds-cake protein, most of them was insoluble in water, but soluble in dilut alkali, and the isoelectric ponit was around pH5.
The results indicated that MW of the most of camellia seeds-cake peptides were below 3000 Da, the oligopeptide(MW below 1000 Da) were predominated (83.73%). The MW of those components was 942.3; the average length of peptide chain(PCL) was 8.4. The oligopeptide was up to 97.54%, the MW changed into 905.1 and PCL to 8.06 after ultrafiltration.
Through three-factor quadratic regression orthogonal rotation experiments, a mathmatical model was set up as follow: Y=-700.499176+30.442000*X1+5.646534*X2-2.555909*X3-0.323605*X1*X1+0.002 000*X1*X2-0.698722*X2*X2+0.236841*X1*X3+0.112727*X2*X3-1.124574*X3*X3
Based on this model, the optimal conditions for ACE inhibitory peptides were: enzymolysis temperature 48.6℃, substrate concentration 4.4%,the ratio of enzyme and substrate concentration 4.2%, pH value7.2,and enzymolysis time 3 hours, ACE inhibitory rate was 46.24%.
The simulation experiments in vitro were used to determine the effects of camellia seeds-cake peptides on scavenging active oxygen free radicals. The results indicated that camellia seeds-cake peptides can scaveng hydroxyl radical, superoxide anion radiacl and DPPH·radical.
引文
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