【作者】 蒋海萍；

【导师】 廖丹葵；

【作者基本信息】 广西大学 ， 化学工艺， 2014， 博士

【摘要】 蓝圆鲹(Decapterus maruadsi)属于鲈形目鲹科圆鲹属的一种,是我国重要的经济鱼类之一。对其加工利用制成鱼糜罐头制品或改性制备功能活性肽,则可大大提高其附加值。本文以蓝圆鲹蛋白为原料,运用酶解技术制各抗氧化肽,采用体外评价法测定其抗氧化活性,通过多种分离技术手段对蓝圆鲹多肽进行分级分离,并采用基质辅助激光解析电离飞行时间串联质谱对目标肽段进行一级氨基酸序列鉴定。新发现的肽段经化学合成后,对其进行体外抗氧化活性验证及反应动力学研究。最后研究了蓝圆鲹多肽在加工储藏过程中,及体外模拟胃肠消化道过程中的抗氧化活性稳定性,并探讨了蓝圆鲹多肽与其他常见抗氧化剂的协同抗氧化效应。主要研究结果如下：(1)通过凯氏定氮法、索氏抽提法和OPA柱前衍生化高效液相色谱法分别测定蓝圆鲹蛋白的基本组分含量。结果表明蓝圆鲹是一种高蛋白(80.16%)、低脂肪(10.38%)、必需氨基酸含量(46.28g/100g protein)和抗氧化性氨基酸含量(32.59g/100g protein)均较理想的食品原料。在此基础上运用蛋白酶解技术,以DPPH-清除活性、O2-··清除活性及还原能力三种体外抗氧化能力为评价指标,分别采用碱性蛋白酶、木瓜蛋白酶、中性蛋白酶、胰蛋白酶和胃蛋白酶五种商业蛋白酶水解蓝圆鯵蛋白。综合比较后选择碱性蛋白酶作为制备蓝圆鲹蛋白抗氧化肽的最佳蛋白酶。(2)以水解度为指标,分别采用单因素法和Box-Benhnken Design (BBD)响应面法优化碱性蛋白酶水解蓝圆鲹蛋白的酶解工艺条件,建立了水解度(Y)与初始底物浓度(S0,X1)、初始酶浓度(E0,X2)、pH (X3)、酶解温度(T,X4)4个因素之间的二元回归模型：Y=22.23-1.05X,+0.66X2+0.79X3+1.09X4-0.082X,X2+0.11X,X3+0.16X,X4-0.17X2X3-0.O67X2X4-0.25X3X4-0.04X12-0.22X22-0.37X32-0.44X42。并根据此数学模型进行参数最优化分析计算得到最佳酶解条件：初始底物浓度16.3g·L-,初始酶浓度0.11g·L-,酶解温度50.5℃, pH8.18,水解300min,理论上可达到22.2%的水解度值,与模型验证实验值22.4%接近。同时,实验研究发现蓝圆鲹蛋白水解产物的抗氧化活性、氨基酸组成及分子量分布均与水解度密切相关。水解度为23.0%的酶解产物的抗氧化活性较高,与其产物中的抗氧化性氨基酸含量相对应。(3)碱性蛋白酶在45.0°C至65.0°C范围内的酶失活热动力学性质研究表明,碱性蛋白酶失活符合蛋白酶一级热失活动力学方程。在50.0℃、pH9.5的反应条件下,碱性蛋白酶酶解蓝圆鲹蛋白的米氏常数Km值为29.115g·L-1。并建立了碱性蛋白酶可控酶解蓝圆鲹蛋白的水解度动力学模型为：水解速率动力学模型为：实验验证表明在一定程度上模型数据与实验结果吻合良好。(4)采用超滤、凝胶层析、高效液相色谱等不同分离纯化技术对蓝圆鲹蛋白酶解产物进行分离纯化,得到抗氧化活性较好的两个高亲水性肽片段B11和B12。采用MALDI-TOF-TOF MS分析鉴定其氨基酸序列结构,得到B11、B12抗氧化肽氨基酸序列分别为His-Asp-His-Pro-Val-Cys (HDHPVC,707.11Da)和His-Glu-Lys-Val-Cys (HEKVC,615.22Da)。B11、B12经化学合成后,分别采用DPPH-清除法、02-·清除法及还原能力法三种体外抗氧化活性检测法验证其抗氧化能力。结果表明两个抗氧化肽均具有较强的抗氧化能力。(5)对抗氧化肽HDHPVC和HEKVC清除DPPH-反应进行了动力学研究。结果表明]HDHPVC和HEKVC均属于慢反应动力学性质,其在固定30min反应时间条件下的ECso值(分别为31.0μM和67.7μM)比稳态条件下的ICso值(分别为9.0μM和14.5μM)偏大3至5倍。HDHPVC和HEKVC的二级反应速率常数k2分别为2.00×10-5μM-’·s-1和0.70×10-5μM-1·S-1,表明HEKVC清除能力较差,而HDHPVC清除能力稍逊于对照物GSH(k2为2.30x10-5μM-1.s-1)。(6)热稳定性实验结果显示,蓝圆鲹多肽(RSH-Ⅲ,Mw<5.0kDa)具有较好的耐热性能；在pH≤7的酸、中性环境中活性保持较好,但不耐碱；蓝圆鯵多肽经体外胃肠道消化后仍能保持70%左右的抗氧化活性,说明其具有一定的耐受胃肠道酶系消化的能力。对蓝圆鲹多肽与其他抗氧化剂的协同抗氧化作用的初探研究发现,只有低浓度条件下的蓝圆鲹多肽与GSH复合清除DPPH·和02-·,及与vitamin C复合清除DPPH·时,表现出了一定的正协同效应,高浓度时存在负协同效应。而当与vitamin C复合清除O2-·时,表现出负协同作用。更多还原

【Abstract】 Round scad (Decapterus maruadsi) belongs to the family of mackerel, it is an important economic fish in China. Processing this low-valued pelagic fish into high market-value products of surimi production and bioactive peptide will pave a way for the full use of round scad. In the study, antioxidative peptide was prepared from round scad protein through enzymatic technology, and purified by a series of separation technology, then matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was used to identify the amino acid sequence of the purified peptides. Subsequently, the identified peptides was synthesized, antioxidative activities and reaction dynamics of the synthetic peptides were studied. At last, the effects of temperature, pH, and the enzyme in the simulated gastrointestinal digestion on the antioxidative peptide were discussed, a pilot study on the synergistic effects between round scad protein and other antioxidants was also carried out. The major results are as follows:(1) The protein content was determined to be80.16%by using Kjeldahl, lipid content of10.38%was measured through Soxhlet extraction method, OPA-precolumn derivatization and RP-HPLC assembly system were used to analyse amino acids composition. Therefore, Round scad is a kind of food material with high protein, low fat, ideal essential amino acids (46.28g/100g protein) and antioxidative amino acids content (32.59g/100g protein). Five commercial proteases, namely, alcalase, papain, neutral protease, trypsin and pepsin were used to hydrolyze round scad protein. The antioxidative activities of round scad protein hydrolysate (RSH) were evaluated through methods of DPPH· scavenging activity, O2-· scavenging activity and reducing power. Therefore, alcalase-treated RSH was selected for further study after comprehensive comparison.(2) Taking the degree of hydrolysis (DH) as criterion, single factor experiment and response surface methodology (RSM) were used to optimize the hydrolysis conditions. The effects of initial substrate concentration (S0, X1), initial enzyme concentration (E0, X2), pH (X3), and temperature (T, X4) on DH (Y) were obtained by multiple regression analysis using Box-Benhnken Design (BBD):Y=22.23-1.05X1+0.66X2+0.79X3+1.09X4-0.082X,X2+0.11X1X3+0.16X1X4-0.17X2X3-0.067X2X4-0.25X3X4-0.04X12-0.22X22-0.37X32-0.44X42. A theoret-ical DH of22.2%could be obtained after reacted for300min under the optimal conditions:initial substrate concentration of16.3g·L-1, initial enzyme concentration of0.11g·L-1,50.5℃and pH8.18, which was validated to be closed to the experimental DH of22.4%. It was also confirmed that the molecular weight distribution, amino acid composition and antioxidative activity of RSH varied with DH. Fractions with the molecular weight under3.0kDa keep increasing with the increase of DH. A slight increase of antioxidative amino acids could be found when DH was ranged from17.0%to23.0%, RSH exhibited the strongest antioxidative activities at23.0%DH.(3) Thermal inactivation of alcalase was found to follow the first-order kinetics in the temperature range from45.0℃to65.0℃. A series of hydrolytic experiments of round scad protein with alcalase were processed at50.0℃and pH9.5. The kinetic parameter, Km, was estimated as29.115g·L-1through Lineweaver-Burk plot. The kinetic equation of DH and hydrolysis rate (r) for the enzymatic hydrolysis of round scad protein by using alcalase were suggested as respectively.(4) Alcalase-treated RSH was purified by ultrafiltration, gel chromatography, and RP-HPLC method, two hydrophilic fractions of B11and B12were obtained. The amino acid sequences of B11and B12were identified as His-Asp-His-Pro-Val-Cys (HDHPVC,707.11Da) and His-Glu-Lys-Val-Cys (HEKVC,615.22Da) by MALDI-TOF/TOF MS, respectively. After being synthesized, antioxidative activities of the two peptides were verified. Results revealed that the two novel peptides exhibited effective antioxidative capacity.(5) Kinetic study of HDHPVC and HEKVC through DPPH-scavenging method indicated that the two peptides could be classified as slow kinetics behaviour. EC50values for HDHPVC and HEKVC at fixed30min reaction time were31.0μM and67.7μM respectively. Whereas, at the steady state, IC50 values for HDHPVC and HEKVC were9.0μM and14.5μM respectively, which were only part of the EC50values at fixed30min. The calculated values of k2for GSH, HDHPVC and HEKVC were2.30×10-5μM-1·s-1,2.00×10-5μM-1·s-1and0.70×10-5μM-1·-s-1, respectively. Thus the antiradical properties could be arranged as GSH≥HDHPVC>HEKVC.(6) The effects of temperature, pH and in vitro simulated digestion model system on the DPPH· and O2-· scavenging activities of RSH-III (Mw<5.0kDa) were investigated. RSH-III exhibited good tolerance for heat treatment and acid conditions under pH7.0, but not alkali-resistant. RSH-III could also resist gastrointestinal enzyme digestion, about70%of initial antiradical activity was maintained after being digested by pepsin and trypsin. A pilot study of synergistic effects between RSH-III and other antioxidants was processed. Among all combinations, group of RSH-III and GSH revealed synergist effects when scavenging DPPH· and O2-· at low concentration. For vitamin C, slight synergist effects could be observed for scavenging DPPH· under low concentration, but there was no synergist effect existed for O2-· scavenging activity.更多还原