【作者】 孙常雁；

【导师】 孔保华；

【作者基本信息】 东北农业大学 ， 农产品加工及贮藏工程， 2014， 博士

【摘要】 乳清蛋白不仅具有良好的营养特性和功能特性,而且美拉德反应产物具有较好的抗氧化功能,近年来大量研究表明乳清蛋白的美拉德反应产物具有较好的抗氧化功能,与合成抗氧化剂相比,具有使用安全的特点,因而在食品应用中受到广泛的关注。但是,目前关于美拉德反应体系的研究中,多集中于蛋白质与还原糖的反应模式,而关于蛋白质水解物与还原糖体系的美拉德反应研究却很少。本试验以乳清蛋白碱性蛋白酶水解物为原料与葡萄糖发生美拉德反应,制备得到乳清蛋白肽美拉德反应产物(Whey protein peptide Maillard reaction products, WPP-MRPs),研究了WPP-MRPs的制备条件、分离纯化工艺及结构分析,并对WPP-MRPs的体外抗氧化活性及诱导肿瘤细胞凋亡作用进行了研究,最后研究了羟基自由基氧化体系(HRGS)对WPP-MRPs结构和功能的影响。论文的研究内容和研究结果如下：1、通过单因素试验和响应面优化试验,以还原力为考察指标,研究了WPP-MRPs的最佳制备工艺条件,结果表明,在葡萄糖浓度为8%、温度为94℃、时间为3.2h条件下制备得到的WPP-MRPs还原力最高,此时还原力(A700nm)达到0.944±0.05。而反应过程中,体系pH值不断降低(P<0.05),褐变程度加深(P<0.05),荧光强度先增加后下降(P<0.05),游离氨基不断减少(P<0.05)。2、以浓度为O.lmg/mL的丁基羟基茴香醚(BHA)和O.lmg/mL的抗坏血酸(Vc)为对照,对WPP-MRPs的抗氧化活性进行了综合评价,结果表明,当WPP-MRPs浓度为40mg/mL时抗氧化能力分别为：还原力(A700nm)为0.39±0.04,羟基清除率为99.37±2.01%,2,2’-氨基-二(3-乙基-苯并噻唑啉磺酸-6)铵盐(ABTS)自由基清除率为81.58±1.33%,Cu2+螯合率为40.47±2.15%, Fe2+螯合率为8.34±0.36%,硫代巴比妥酸反应物质(Thiobarbituric acid-reactive substances, TBARS)值为1.13±0.07mg/L。此浓度的WPP-MRPs羟基清除率和金属螯合力(Cu2+, Fe2+)显著高于Vc和BHA(P<0.05);而还原力显著低于Vc(P<0.05),但与BHA相比差异不显著(P>0.05); ABTS自由基清除率略低于Vc和BHA; TBARS抑制作用低于BHA但好于Vc。3、试验研究了温度、pH值、光照、金属离子和过氧化氢对WPP-MRPs抗氧化稳定性的影响。结果表明,WPP-MRPs的抗氧化活性受热影响较小,在50～110℃下加热时还原力、羟基和ABTS自由基清除率变化差异不显著(P>0.05)；在偏碱性环境中的抗氧化活性大于偏酸性环境中,pH值为8.0时还原力、羟基和ABTS自由基清除率最高(P<0.05)；在不同光照条件下放置25d, WPP-MRPs抗氧化活性均显著下降(P<0.05),其中在室外自然光条件下降低最显著(P<0.05),其次是室内自然光和避光；金属离子Na+、K+、Mg2+、Ca2+、Zn2+对WPP-MRPs的还原力、羟基和ABTS自由基清除率的影响不显著(P>0.05),而金属离子Cu2+、Fe2+、Fe3+和氧化剂H202能够显著降低WPP-MRPs的抗氧化活性(P<0.05)。因此,在加工、储藏过程中应注意WPP-MRPs在偏碱、避光及避开铜铁器具和H202条件下使用,这样可以发挥其良好的抗氧化作用。4、试验采用中空纤维超滤膜对WPP-MRPs进行分级得到Ⅰ(分子量<0.8kD)、Ⅱ(0.8kD<分子量<51dD)、Ⅲ(分子量>5kD),对还原力最强的Ⅱ(A700nm为1.55±0.06)再进行凝胶过滤色谱纯化,最佳凝胶纯化条件为：以Sephadex G-25为纯化介质,采用1.0x50cm纯化柱,择蒸馏水为洗脱液,缓冲液流速为0.2mL/min,进样量为1.5mL,在该条件下进行分离纯化后得到三个不同分子量范围的组分WM,(2780.75～7436.78D)、WM2(813.08-2780.75D)和WM3(54.36-813.08D)。经过抗氧化活性研究得出WM2抗氧化性较好(P<0.05),在浓度40mg/mL时WM2的还原力(A700nm)为0.68±0.04,对羟基自由基清除能力99.95±±2.70%,ABTS自由基清除率为88.63±3.04%,Cu2+螯合率为45.41±1.12%,Fe2+螯合率为11.28±±0.05%,TBARS值为0.83±0.06mg/L.5、结构分析结果表明,WPP-MRPs的赖氨酸含量显著下降(P<0.05),较乳清蛋白肽(WPP)的赖氨酸含量降低了0.86%,精氨酸、组氨酸及脯氨酸含量也有一定程度下降(P<0.05)；傅立叶变换红外光谱(FTIR)和圆二色光谱(CD)分析表明,糖分子连接到乳清蛋白肽分子上,使WPP的二级结构中α螺旋和p转角的含量减少(P<0.05),β折叠和无规则卷曲的含量增加(P<0.05),说明美拉德反应使WPP的二级结构由有序向无序转变；荧光光谱分析表明,在420～430nm处出现新的吸收峰,说明产物生成了具有荧光性的生色团物质；紫外吸收光谱扫描结果表明,WPP经过美拉德反应后在220nm和280nm处紫外吸收强度增加,说明生色团物质含量增加。6、试验研究了WM2对宫颈癌细胞Hela及结肠癌细胞Ht-29的抑制作用,结果表明,WM2能够以剂量依赖和时间依赖的方式抑制癌细胞的增殖,作用12h和24h后对Hela细胞的IC50分别为39.12mg/mL和35.91mg/mL,对Ht-29细胞的IC50分别为35.80mg/mL和31.94mg/mL;通过倒置显微镜观察细胞结构,发现经WM2作用以后,细胞出现典型的凋亡特征,细胞收缩,胞质浓缩,形成胞浆丝,细胞失去贴壁能力；流式细胞检测结果进一步说明了WM2能够诱导Ht-29细胞发生凋亡。WM2抗氧化活性与Ht-29抑制率的相关性分析表明,对癌细胞抑制率与羟基清除率的相关系数为0.91(P<0.05),与ABTS自由基清除率的相关系数为0.90(P<0.05),与还原力的相关系数为0.94(P<0.05)。7、试验研究了HRGS对WM2结构和功能的影响,结果表明,随着H202浓度的增加和氧化时间的延长,WM2中的二聚酪氨酸含量显著上升(P<0.05),活性巯基、游离氨基含量均显著下降(P<0.05); WM2的乳化功能受到影响,在0.1mmol/L的H202氧化1h后乳化性和稳定性略有上升(P>0.05),随着H202的浓度增加和氧化时间的延长乳化性和稳定性又显著下降(P<0.05)。本论文证明了WPP-MRPs及其分离纯化产物具有较强的抗氧化活性,而且稳定性良好,适合各类食品加工,因此可以广泛用于食品、保健品中。因此本研究为拓宽乳清蛋白的应用及开发天然安全的抗氧化剂提供了依据和思路。更多还原

【Abstract】 Whey protein has drawn widely attention in food industry for its high nutritional value and excellent functional properties. Some studies suggested that the Maillard reaction products (MRPs) formed from whey protein and sugars have a better antioxidatant activity. Compared with synthetic antioxidants, natural antioxidant has a higher safety. Recently, the most of studies about the antioxidant activity of MRPs focus on the reaction between proteins and reducing sugar, and few studies involve the antioxidant activity of MRPs from whey protein hydrolysates and reducing sugar.In this study, Maillard reaction products of whey protein peptide and reducing sugar (WPP-MRPs) were obtained by the reaction between alcalase hydrolysates products of whey protein and glucose. First, the preparation conditions of WPP-MRPs were determined. Its structures after separation and purification were analyzed, and its antioxidant activity in vitro and effect on inducing tumor cell apoptosis were evaluated. Finally, the influences of the hydroxyl radical-generating system (HRGS) on the structure and function of WPP-MRPs were studied. The main results are as follows:1. The optimum preparation conditions of WPP-MRPs were established by single factor experiment and response surface optimization design, and the reducing power were used as analyzing index. The optimum preparation parameters to obtain the highest reducing power were8%glucose concentration at94℃for3.2h, and the reducing power (A700nm) was0.94±0.05. During reaction, the pH value in solution constantly decreased (P<0.05), the browning degree deepened (P<0.05), fluorescence intensity experienced the trend of rising at first and then falling (P<0.05), and the free amino acid gradually decreased (P<0.05).2. The antioxidant activities of WPP-MRPs were evaluated, and butyl hydroxy anisole (BHA;0.1mg/mL) and ascorbic acid (Vc;0.1mg/mL) were used as control. The results showed when the concentration of WPP-MRPs was at40mg/mL, the reducing power, hydroxyl radical scavenging rate, ABTS radical scavenging rate, and Cu2+chelating ability were0.39±0.04,99.37±2.01%,81.58±1.33%, and40.47±2.15%, respectively. Compared with0.1mg/mL Vc and0.1mg/mL BHA,40mg/mL WPP-MRPs exhibited a significant higher hydroxyl radical scavenging ability and a stronger metal (Cu2+, Fe2+) chelating ability (P<0.05), while its reducing power was lower than Vc (P<0.05), and has no significantly difference compare to BHA. The inhibition of WPP-MRPs to TB ARS (thiobarbituric acid-reactive substances) was also lower than BHA and higher than Vc.3. The antioxidant stabilities of WPP-MRPs were studied by evaluating the effects of temperature, pH, light condition, metal ions and H2O2on the antioxidant activity. The results showed that heating has a little effect on the antioxidant activity of WPP-MRPs. The total reducing power, hydroxyl radical scavenging rate and the ABTS radical scavenging rate of WPP-MRPs had no significant changes when WPP-MRPs were heated at50～110℃(P>0.05). The antioxidant activity of WPP-MRPs at slightly alkaline condition was stronger than that at slightly acidic or neutral condition, and the total reducing power, hydroxyl radical scavenging rate and the ABTS radical scavenging rate of WPP-MRPs were the highest at pH8.0(P<0.05). The WPP-MRPs were placed under the condition of outdoor natural light, indoor natural light and avoid light for25d. The results showed that the antioxidant activity of WPP-MRPs had significantly decreased at outdoor natural light and the decreasing order was:outdoor natural light> indoor natural light> avoid light (P<0.05). Metal ions, such as Na+, K+, Mg2+, Ca2+and Zn2+, had few effects on the total reducing power, the hydroxyl radical and the ABTS radical scavenging ability of WPP-MRPs (P>0.05). However, the metal ions such as Cu2+, Fe2+, Fe3+and the oxidant H2O2could largely reduce the antioxidant activity of WPP-MRPs (P<0.05). So, WPP-MRPs should be processed at conditions of slightly alkaline, stored at dark, and avoided contact copper and iron utensils and H2O2in order to keep its good antioxidant.4. WPP-MRPs were classified through the hollow fiber ultrafiltration membrane, and part I (molecular weight<0.8kD), Ⅱ(0.8KD<molecular weight<5kD), and III (molecular weight>5kD) were obtained. Part Ⅱ had the best reducing power (P<0.05) and was used for gel filtration chromatography purification. The optimal purification condition for preparing WM (WPP-MRPs II)was determined by single factor experiment by using Sephadex G-25column (1.0×50cm) as the purification medium and the distilled as the eluant. Three components with different molecular weight ranges, including WM1(2781-7437D), WM2(813-2781D), and WM3(54-813D) were obtained after separation and purification under the conditions of buffer flow rate at0.2mL/min and the injection volume at1.5mL. The results showed that WM2had better antioxidant activity (P <0.05). When the concentration of WM2was40mg/mL, its reducing power (A700nm) was0.68±0.04; the hydroxyl radical scavenging rate was99.95±2.70%; the ABTS radical scavenging rate was88.63±3.04%; Cu2+chelating ability was45.41±1.12%; Fe2+chelating ability was11.28±0.05%; and TBARS was0.83±0.06mg/L.5. The structure analysis showed that the total amino acid composition of the WPP-MRPs experienced significant changes. Compared with that of WPP, lysine content declined significantly and decreased by0.86%, and the contents of arginine, histidine and proline experienced slightly declining as well (P<0.05). Fourior transform infrared spectroscopy (FTIR) analysis and circular dichroism (CD) indicated that saccharide conjugated with WPP covalently, which made the content of a-helical and β-turn in the secondary structure of MRPs decreased (P<0.05), but that of (3-sheet and random structure increased (P<0.05). This revealed that Maillard reaction had a certain effect on the secondary structure of WPP. It made the transition from order to disorder. The results of circular dichroism (CD) spectra correspond with that of FTIR in spite of only slight difference in data. Fluorescence spectrum showed that a new peak value occurred between420nm and430nm, indicating that a kind of new substance with fluorescence property emerged in the MRPs. The scan results of ultraviolet absorption spectrum suggested that Maillard reaction increased the chromophore content.6. The inhibition of WM2on cervical cancer cells (Hela) and colon cancer cells (Ht-29) were studied, the result indicated that WM2inhibited the proliferation of Ht-29and Hela cells dependent on dose and time. The IC50value of Hela cells were39.12mg/mL和35.91mg/mL, respectively, through interacting about12hours and24hours, and the figures of Ht-29cell’s IC50were35.80mg/mL and31.94mg/mL. The cell expressed typically apoptosis characteristic such as cell shrinkage, formation of cytoplasmic filaments, condensation of nuclear chromatin, and loss of cell adherent capacity after interacting with WM2by observing the cell structure by the inverted microscope. Furthermore, flow cytometry results further illustrated the WM2induced Ht-29cell apoptosis. Correlation analysis between WM2antioxidant activity and Ht-29inhibition rate showed the correlation coefficient of cancer cell inhibition rate with hydroxy clearance rate was0.91(P<0.05), and the correlation coefficient with ABTS radical scavenging was0.90(P<0.05), and the correlation coefficient with reducing power was0.94(P<0.05).7. The effects of HRGS on WM2structure and function of WM2were studied. The results indicated that the dimer of L-tyrosine of WM2after oxidation exhibited a significant increase with increased H2O2concentration and oxidation time (P<0.05). Active thiol and free amino were both apparently declined (P<0.05). The oxidation made the structure of WM2change, which impacted its emulsifying function. Studies demonstrated that the emulsifying property and stability of WM2rose a little after O.lmmol/L of H2O2oxidation for1h (P>0.05), but the emulsifying property and stability of WM2significantly decreased with ascending concentrations of H2O2and extending the oxidation time (P<0.05).In conclusion, WPP-MRPs and their separation and purification products possess strong antioxidant activity and appropriate stability. Therefore, they can be extensively utilized in foods and health-care products. Besides, this research provides basis and ideas for expanding whey protein modification and developing natural and efficient antioxidants as well.更多还原