【作者】 何思莲；

【导师】 宁正祥；

【作者基本信息】 华南理工大学 ， 食品科学， 2013， 博士

【摘要】 本文以鳀鱼蛋白为原料，研究了不同的内源酶水解条件对酶解产物抗氧化活性的影响，确定了鳀鱼蛋白内源酶水解制备抗氧化肽的最佳工艺条件；考虑内源酶失活、底物和产物抑制对内源酶水解的影响，推导和建立水解度和反应时间的动力学经验方程。在内源酶水解的基础上添加外源性商品酶协助内源酶水解，确定了最佳用酶和最优水解工艺条件。通过采用体外抗氧化试验，综合地评价了鳀鱼蛋白酶解物的抗氧化效果；运用现代分离纯化和氨基酸测序技术对鳀鱼蛋白酶解产物的抗氧化肽进行分离纯化和鉴定；研究了鳀鱼蛋白酶解产物的理化性质和抗氧化稳定性。鳀鱼蛋白含量15.7%，氨基酸组成丰富，脯氨酸、亮氨酸，缬氨酸和芳香性氨基酸的含量为32.0%。底物浓度为42.4mg/mL、温度50℃、pH7.5、内源酶水解时间3h获得的水解产物DPPH自由基清除活性、水解度和总氮回收率分别为64.2%，24.3%和77.0%。推导并验证了鳀鱼蛋白内源酶水解动力学模型，内源酶水解体系动力学和热力学常数分别为Km=8.7g/L、k2=1.4gU-1min-1、Ks=73.9g/L和kd=0.15gU-1min-1，△EA=2.68×104J/mol，体系中存在底物抑制。当采用外源酶协助内源酶水解时，最优的用酶和水解条件为：最佳的商品蛋白酶组成：Protamex：Flavourzyme500MG：Alcalase2.4L=1.1:1.0:0.9，E/S为3.27%（w/w，protein），水解时间2.7和温度55.4℃，该条件下获得的水解产物DPPH自由基清除活性、水解度和总氮回收率分别为84.7%，33.2%和87.5%。抗氧化活性、水解度、总氮回收率比内源酶水解产物分别提高了20.5%，8.9%和10.5%。内源酶水解产物（APH）和最佳商品酶协助内源酶水解的产物（CAPH）均表现出良好的的抗氧化活性，其中CAPH的还原力、DPPH自由基清除能力、羟基自由基清除能力、超氧阴离子自由基清除能力和螯合金属离子能力优于APH，且抗氧化能力与浓度有一定的量效关系。鳀鱼蛋白水解产物的亚油酸自氧化抑制能力与BHT相比较低，APH的亚油酸自氧化抑制能力稍强于CAPH。通过超滤、凝胶过滤色谱和RP-HPLC对鳀鱼蛋白酶解产物分离纯化，最后以基质辅助激光解吸电离飞行时间质谱法(MALDI-TOF-TOF MS)鉴定出该肽段的氨基酸序列为Ser-Gln-Phe-Pro-Phe-His-Ala-Arg-Gln-Gln，分子量1244.8Da。鳀鱼蛋白酶解产物的必需氨基酸（EAA）含量均满足世界卫生组织/联合国粮食及农业组织（FAO/WHO）模式（1991）。APH和ACPH的鲜味氨基酸含量分别为44.83%和44.59%，在游离氨基酸中，APH和CAPH的鲜味氨基酸含量分别为9.46%、12.42%，商品酶协助内源酶水解更有利于鲜味氨基酸以游离的形式释放。在AHP和CAPH中肽的质量百分数分别为67.15%、62.93%，主要为分子量小于1kDa的小肽，分子量低于5kDa的肽的百分含量分别为90.1%、96.2%。在pH2.0-10.0范围内，鳀鱼蛋白水解产物的溶解性均在80%以上，溶解性在pH4.0时降到最低，当pH8.0时最高分别可达97.3、98.9%。pH﹤5.0时，鳀鱼水解产物的浊度较高，并在pH3.0（CAPH）或pH4.0（APH）时浊度达到最大。当pH≥4.0时，浊度明显降低。在pH2.0-10.0范围内，鳀鱼水解产物的抗氧化活性可保持在80%以上。在20-100℃范围内，APH和CAPH的抗氧化活性变化不显著（P>0.05），100℃下处理2h，抗氧化活性保持率仍在96%以上。经体外模拟胃肠道消化酶消化后，APH和CAPH的抗氧化活性保持率分别为72.3%，70.5%，比消化前分别下降了27.73%，29.5%。更多还原

【Abstract】 The present study deals with the effects of different conditions on the antioxidantactivity of protein hydrolysates of anchovy autolysis. The optimum conditions weredetermined to obtain high antioxidant activity hydrolysate. Considering the effects ofinactivation of enzyme, inhibition of substrate and product inhibition on the autolysis, aempirical equation autolysis kinetics about the relationship between DH and autolysis timewas established. On the basis of the autolysis exogenous enzymes was added to obtainhydrolysate with higher antioxidant activity. The best enzyme and the optimal hydrolysisconditions were determined. The antioxidant effect of the anchovy protein hydrolysates wascomprehensive evaluated by using vitro antioxidant tests. Antioxidant peptides from anchovyprotein hydrolysates were isolated and identified using modern purification and amino acidsequencing technology. The physicochemical properties and oxidation stability of anchovyprotein hydrolysate was studied.The protein content of anchovy was15.7%, rich in amino acids. The total content ofproline, leucine, valine and aromatic amino acid content was32.0%. At the condition ofsubstrate concentration42.4mg/mL, temperature50°C, pH7.5and autolysis time3hhydrolyzate obtained DPPH radical scavenging activity, the degree of hydrolysis and totalrecoveries were respectively64.2%,24.3%and77.0%. The anchovy protein autolysisdynamics model of was derived and validated. Autolysis dynamics and thermodynamicsconstants were Km=8.7g/L、k2=1.4gU-1min-1、Ks=73.9g/L、kd=0.15gU-1min-1，△EA=2.68×104J/mol. There is substrate inhibition phenomenon in the process of autolysis.When using exogenous enzymes to assist autolysis, the optimal enzyme and hydrolysisconditions were as follow: Best of goods protease composition: Protamex:Flavourzyme500MG: Alcalase2.4L=1.1:1.0:0.9, E/S of3.27%(w/w, protein),2.7hydrolysis time and temperature55.4°C. Under this conditions the DPPH radical scavengingactivity, DH and total nitrogen recovery of hydrolyzate were84.7%,33.2%and87.5%,respectively, which were respectively increased by20.5%,8.9%and10.5%compared withautolysate.Autolysate (APH) and hydrolysate prepared by best commercial enzyme assisted autolysis (CAPH) showed good antioxidant activity, wherein the reducing power, DPPHradical scavenging activity, hydroxyl radical scavenging activity, superoxide anion radicalscavenging activity and chelating metal ions of CAPH was better than that of APH. Theantioxidant activities have concentration-effect relationship. Antioxidant activities of anchovyprotein hydrolysate in linoleieaeid system were low compared with BHT, Inhibition activityof APH is slightly stronger than that of CAPH.Antioxidant peptide was purified from anchovy protein hydrolysates by ultrafiltration,gel filtration chromatography and RP-HPLC. The amino acid sequence of peptide isidentified as Ser-Gln-Phe-Pro-Phe-His-Ala-Arg-Gln-Gln by matrix-assisted laserdesorption/ionization time-of-flight mass (MALDI-TOF-TOF MS). The molecular weight ofpeptide is1244.8Da.Essential amino acids (EAA) content of anchovy protein hydrolysates of were metWorld Health Organization/Food and Agriculture Organization (FAO/WHO) mode (1991).The flavor amino acid of APH and ACPH f were44.83%and44.59%, respectively. In freeamino acids, flavor amino acid contents of APH and CAPH were9.46%,12.42%,respectively. Commercial enzyme assited autolysis is more conducive to the relaease of flavoramino acid in the form of free amino acid. The mass percentage of peptide of AHP andCAPH were67.15%,62.93%, respectively. The molecular weight of most peptides is lessthan1kDa. The percentage content of peptide with molecular weight less than5kDa of AHPand CAPH were90.1%,96.2%, respectively.In the range of pH2.0-10.0, solubility of anchovy protein hydrolyzate was above80%.The minimum solubility was at pH4.0. When pH was8.0, the solubility of APH and CAPHwere respectively up to97.3and98.9%. When pH <5.0, the turbidity of anchovy hydrolysatewas high and the maximum turbidity appeared at pH3.0(CAPH) or pH4.0(APH). When pH≥4.0, the turbidity was significantly reduced.In the range of pH2.0-10.0, anchovy hydrolyzate antioxidant activity can be maintainedat80%. At20-100°C range, APH and CAPH antioxidant activity did not change significantly(P>0.05). After2h treatment at100°C, antioxidant activity was still maintained at above96%. After digestion by in vitro gastrointestinal enzymes, APH and CAPH antioxidant activity retention rates were respectively72.3%and70.5%, decreased by27.73%,29.5%compared with that before digestion.更多还原