【作者】 王可；

【导师】 林松毅；

【作者基本信息】 吉林大学 ， 食品科学， 2014， 硕士

【摘要】 本论文研究内容隶属于“十二五”国家科技支撑计划课题《食源性功能肽生物制备技术研究（2012BAD33B03）》，是以蛋白质含量为79.4%、粒径为180目的玉米蛋白粉为原料，重点开展了高效水解玉米蛋白粉的关键技术、高活性玉米抗氧化肽（CAPS）的优选、高活性玉米抗氧化肽（CAPS-1）的安全毒理学评价、玉米抗氧化肽（CAPS-1）分离制备高活性因子、基于高压脉冲电场技术（PEF）提高玉米抗氧化肽活性等研究工作，具体研究内容如下：（1）在高效水解玉米蛋白粉的关键技术研究中，以水解度（DH）为衡量指标，借助单因素实验和响应面实验设计等研究方法，对比分析了碱性蛋白酶、中性蛋白酶和木瓜蛋白酶3种蛋白酶对玉米蛋白粉酶解过程中的DH的影响情况。通过对酶解温度T、酶解pH值、酶比底物浓度[E]/[S]的单因素实验对比分析获知，碱性蛋白酶酶解制备玉米蛋白粉酶解物的DH最高。通过响应面实验设计，构建了碱性蛋白酶回归模型为Y=36.85333-1.48375X1-9.94125X2+1.12X3-0.5225X1X2+0.78X1X3-0.31X2X3+0.024583X12-11.8954X22-1.74292X32，式中：X1为酶解温度（℃）；X2为酶解pH值；X3为酶比底物浓度[E]/[S（]%）。其最佳工艺参数为：酶解温度50℃，酶解pH值8.6，酶比底物浓度9.13%，玉米蛋白粉的最大水解度可达40.26±0.23%；在此基础上探讨了辐照剂量（0-138.7kGy）对玉米蛋白粉酶解过程中DH的影响，获知当辐照剂量为65.0kGy时，玉米蛋白粉的DH可达最高值48.23±6.1%，经场发射扫描电镜检测辐照前后玉米蛋白粉表面形态变化，获知较未辐照玉米蛋白粉相比，辐照剂量为65.0kGy的玉米蛋白粉表面的凹凸程度增加，完整性下降。（2）在高活性玉米抗氧化肽（CAPS）的优选的研究中，以最佳酶解工艺所获得的玉米蛋白酶解物为原料，以经超滤技术制备获得的CAPS-1，CAPS-2，CAPS-3，CAPS-4和CAPS-5五种不同分子量范围的超滤液为研究对象，借助于体外抗氧化评价指标（DPPH清除率）和体内抗氧化动物实验指标（受试小鼠血清中的GSH-Px活力、CAT活力和MDA含量），优选出抗氧化活性最高的超滤液组分。研究发现：体外抗氧化评价结果表明CAPS-1的DPPH清除率可达最高值84.75±4.19%。体内抗氧化动物实验结果表明：与空白组相比，CAPS-1-中剂量（CAPS-1-MD）组的GSH-Px活力显著增加（P<0.05），可达最高值114.09±5.4U/mL； CAPS-1-MD组的CAT活力显著增加（P<0.05），可达最高值14.18±0.58U/mL；CAPS-1-MD组的MDA含量显著降低（P<0.05），降至15.35±3.09U/mL。（3）在高活性玉米抗氧化肽（CAPS-1）的安全毒理学评价研究中，是以超滤分离获得的CAPS-1为实验原料，选取受试小鼠的精子畸形率、骨髓微核率、NK细胞活性、脏器系数（心脏系数、肺系数、肾脏系数、肝脏系数和胸腺系数）、体重均值为衡量指标，研究结果表明：与空白组相比，CAPS-1对受试小鼠的精子畸形率、骨髓微核率、脏器系数（心脏系数、肺系数、肾脏系数、肝脏系数、胸腺系数）和体重均值的影响均不显著（P>0.05）；CAPS-1可以使受试小鼠的NK细胞活性显著提高（P<0.05），最高可达205.1±19.48%，较空白组提高44.09%。（4）在玉米抗氧化肽（CAPS-1）分离制备高活性因子的研究中，以CAPS-1为实验原料，以DPPH清除率为衡量指标，利用Sephadex G-25凝胶色谱分离技术为分离纯化方法，当Sephadex G-25凝胶色谱分离装置的层析柱径高比为1.6:100、上样浓度为50mg/mL、洗脱流速为60mL/h时，分离获得CAPS-1-Ⅰ、CAPS-1-Ⅱ、CAPS-1-Ⅲ和CAPS-1-Ⅳ4个组分，研究发现CAPS-1-Ⅲ的DPPH清除率可达最高值84.51±1.3%；将CAPS-1-Ⅲ进行氨基酸成分分析获知，与CAPS-1相比，CAPS-1-Ⅲ中Glu和Leu的含量显著增加，分别由1.581%增至3.237%，由1.801%增至2.689%，疏水性氨基酸比例降低，降低率为13.3%。（5）在基于高压脉冲电场技术（PEF）提高玉米抗氧化肽活性的研究中，以CAPS-2和蛋清抗氧化肽（EWAPS）为实验原料，以DPPH清除率为衡量指标，借助单因素和响应面实验设计方法，构建了PEF技术提高CAPS-2抗氧化活性的工程化回归模型为Y=-152.29-4.57X1+7.93X2+0.18X3+0.01X1X2-0.002X1X3-0.002X2X3+0.53X12-0.13X22-0.00003X32，式中X1为料液浓度（mg/mL）、X2为电场强度（kV/cm）、X3为电场频率（Hz）；最佳技术参数为：料液浓度6mg/mL、电场强度11.11kV/cm、电场频率2333.57Hz，此条件下PEF处理的CAPS-2的DPPH清除率可达到86.88±0.41%，较处理前提高了21.3%。更多还原

【Abstract】 Key technologies of efficient utilization of Corn gluten meal to develop antioxidativepeptides were granted by the National Science and Technology Pillar Program during the12thFive-year Plan Period (No.2012BAD33B03). Corn gluten meal (protein content of79.4%,sieving mesh of180) were used as raw material. The key technologies of high efficienthydrolysis of Corn gluten meal, optimization of Corn antioxidant peptides (CAPS) with highactivity, toxicological safety evaluation of CAPS with high activity, preparation of high active ofCAPS, and the technology of pulsed electric field (PEF) improving the activity of CAPS wereresearched. The specific research content as follows:(1) The efficient hydrolysis technologies of Corn gluten meal were researched. Alacalse,Neutral protease and Papain were used to hydrolyze corn protein, and degree of hydrolysis (DH)was used as indexs. One-factor-at-a-time (OFAT) and response surface metrology (RSM)experiment were used to optimize the hydrolyze process. The results of hydrolysis process of3enzymes were obtained, and it showed that Alcalse was optimized for the efficient enzyme ofCorn gluten meal. The final equation in terms of actual factors was:Y=36.85333-1.48375X1-9.94125X2+1.12X3-0.5225X1X2+0.78X1X3-0.31X2X3+0.024583X12-11.8954X22-1.74292X32，in the equation：X1was enzyme solution temperature (℃)；X2was pHvalue；X3was [E]/[S](%). The optimized processing parameters were: temperature50℃, pH8.6,[E]/[S]9.13%, the predicted value of DH was40.26±0.23%. Then, irradiation doses(0-138.7kGy) were used for further improvement of the DH. The influence of differentirradiation dose DH of Corn protein was discussed. The optimal irradiation dose (65.0kGy) wasselected by OFAT experiment. The DH of the Corn gluen meal was48.23±6.1%. And themicrocosmic surface features of material were analyzed by scanning electron microscope test.The results showed that, the particle surface of Corn gluten meal powder with irradiationincreased, compared with blank sample.(2) The Corn protein hydrolysate prepared with optimal hydrolysis process was separatedby ultrafiltration. Different fractions of Corn antioxidant peptides (CAPS) were compared byantioxidant capacity in vivo and in vitro. The results indicated that CAPS-1had the highest antioxidant capacity. The DPPH inhibition of CAPS-1was84.75±4.19%. And GSH-Pxcapacity of CAPS-1-MD was114.09±5.4U/mL, the CAT capacity of CAPS-1-MD was14.18±0.58U/mL, and the MDA content of CAPS-1-MD was15.35±3.09U/mL.(3) Toxicity and safety evaluation of CAPS-1obtained by ultrafiltration were researched.The sperm deformity rate, micronucleus rate, NK cell activity and organ coefficients were tested.The results indicated that, CAPS-1group had no significant toxic harm in mice, and had certainimmune activities.(4) CAPS-1was separated by Sephadex G-25purification. The diameter to height ratio ofchromatography column was1.6:100, sampling amount was50mg/mL, and flow was60mL/h.DPPH inhibition test was used to evaluate the oxidation capacity of the fraction collected. Theresults showed that, CAPS-1-Ⅲ had highest antioxidant activity, with DPPH inhibition of84.51±1.3%. And the results of amino acid analysis showed that, the content of Glu and Leu inpurified Corn antioxidative peptides were significantly increased, the content of hydrophobicamino acids were decreased.(5) Pulsed electric field (PEF) was used to improve the activity of CAPS-2and EWAPS.OFAT and RSM test were used to optimaze technological parameters of PEF. The final equationin terms of actual factors was: Y=-152.29-4.57X1+7.93X2+0.18X3+0.01X1X2-0.002X1X3-0.002X2X3+0.53X12-0.13X22-0.00003X32, in the equation：X1was concentration of CAPS-2(mg/mL);X2was electric strength (strength)；X3was frequency (Hz). And the optimizedprocessing parameters were: concentration6mg/mL, electric field strength11.11kV/cm,and frequency2333.57Hz. In this condition, DPPH inhibition was86.88±0.41%, was increasedby21.3%compared with untreated sample.更多还原