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猪血中超氧化物歧化酶(SOD)和血红素的提取及SOD的应用研究

Extraction of Superoxide Dismutase(SOD) and Heme from Porcine and the Application of SOD

作者: 专业:食品科学 导师:李小定 年度:2010  院校: 华中农业大学

Keywords

        我国猪血资源非常丰富,猪血的蛋白质含量高、氨基酸组成平衡,是优质的蛋白来源。猪血中以红细胞为主,红细胞中最主要的蛋白是血红蛋白,含量占血细胞蛋白总量的90%以上。开发猪血资源不仅可增加蛋白质饲料来源、增加用于食品、医学等领域的原料资源,又可减少屠宰废弃物对环境的污染,具有很好的经济和社会效益。本论文以猪血为原料提取SOD和血红素,进行初步鉴定、储存过程中的稳定性实验,并对SOD的体外抗氧化活性及其对肿瘤细胞凋亡的影响进行研究。1 SOD和血红素提取工艺条件的优化采用超声波破碎细胞膜,SOD比活力和血红素提取率都较高的超声条件为:超声功率70 W、超声时间15 min及超声温度40℃;利用Box-Behnken的中心组合旋转设计试验,Design-Expert软件处理数据,得出SOD的最佳提取条件:热变温度65.2℃、热变时间26.1 min、0.1 mol/L氯化铜添加0.423mL/15mL溶血液;血红素的最佳提取条件为:酸性丙酮添加量为血红蛋白的5.66倍,酸酮比为3%,抽提时间为42.00 min时血红素提取率达到最大值为0.225 g/100 mL。2 SOD和血红素的初步鉴定SOD提取样品的紫外光谱分析最大吸收峰为265 nm,与文献报道一致;血红素标品和样品进行紫外光谱、红外图谱和高效液相色谱分析确定提取物为血红素。选择甲醇:水为80:20,在所选流动相和仪器条件下,检出限、精密度和稳定性均良好,血红素的纯度为72%。3 SOD和血红素的储存稳定性研究SOD和血红素在储存过程中,冷冻保存有利于SOD和血红素的稳定。储存20d时,冷冻保存SOD的相对活力为90%,冷藏保存相对活力为85%,室温避光保存为82%,室温光照相对活力为80.25%;储存9周后,冷冻保存的血红素相对含量为91.58%,冷藏保存、室温避光保存和室温光照保存的相对含量分别为88.61%、83.46%和81.75%。4 SOD的体外抗氧化活性及对小鼠荷瘤sp2/0细胞凋亡的影响SOD的体外抗氧化实验中以Vc作为对照品,检测SOD的体外抗氧化活性,由实验结果可知SOD有一定的抗氧化活性,但其抗氧化活性比同浓度的Vc低。采用研究细胞凋亡的最基本的方法:凋亡细胞的形态学观察。由本实验可见未加SOD的小鼠荷瘤sp2/0细胞生长良好,表现为圆形;经不同浓度SOD作用一段时间后的小鼠荷瘤sp2/0细胞其细胞核明显固缩、碎裂,表现为凋亡形态,且随着作用浓度的增加,细胞核固缩碎裂的程度增加。SOD在体外可在一定程度上抑制小鼠荷瘤sp2/0细胞的增殖,呈时间-效应和剂量-效应的关系,能够诱导小鼠荷瘤sp2/0细胞的凋亡。
    Pig blood is very rich in our country, it is rich in protein and amino acid is balance, it is the source of high-quality protein. Red blood cell is the principal components in pig blood, the main protein in red blood cell is hemoglobin, account for more than 90%. To develop pig blood resources has good economic and social benefits, it is not only can increase the protein of feed sources, increase raw material resources for food, medicine and other fields, but also to reduce the pollution of environment from animal waste. In this thesis, SOD and heme were extracted from pig blood, structural identification, stability in stored procedure of SOD and heme, the in vitro antioxidant activity and the effect on mouse-borne tumor cell apoptosis of SOD were studied.1 The optimization extraction condition of SOD and hemeWhen broken the cell membrane by ultrasonic, the optimum ultrasonic conditions for high SOD specific activity and high heme extraction rate were:ultrasonic power 70 W, ultrasonic time 15min and ultrasonic temperature 40℃; Using Box-Behnken of the central composite rotary design test, Design-Expert software to process data, the best extraction conditions obtained SOD:heating temperature 65.2℃, heating time 26.1 min, copper chloride solution of 0.1 mol/L added 0.423 mL/15 mL dissolve blood; the optimum extraction conditions of heme:acidic acetone addition level was 5.66 times that of hemoglobin, ratio of hydrochloric acid and acetone was 3%, the extraction time was 42.00 min, the maximum extraction rate of heme was 0.225 g/100 mL.2 Initial structure identification of SOD and hemeAnalyzed extracted SOD samples by UV spectrum, the maximum absorption peak is 265 nm, the result was consistent with those reported; compared the spectrogram of standard heme and samples heme by UV spectrum, Infrared spectrum and high performance liquid chromatography, the extraction sample was proved to be heme. Under the choosen mobile phase(methanol:water was 80:20) and equipment condition, detection limits, precision and stability was good, purity of heme was 72%. 3 Storage stability of SOD and hemeFreezing storage was conducive to the stability of SOD and heme in storage. In freezing storage, chill storage, room temperature and dark storage, room temperature in daylight storage, the relatively activity of SOD were 90%,85%,82%and 80.25%after 20 h. In freezing storage, chill storage, room temperature and dark storage, room temperature in daylight storage, the relatively contents of heme were 91.58%,88.61%,83.46% and 81.75% after 9 weeks.4 Antioxidant activity and effects on mouse-borne tumor sp2/0 cell apoptosis of SODThe antioxidant activity of SOD was detected in vitro, using Vc as the reference standard, the results showed that SOD had good antioxidant activity, but lower than that of Vc.Observation of cell morphology is the basic method of apoptosis study. It was indicated that mouse-borne tumor SP2/0 cell grew well without SOD, showing round; mouse-borne tumor SP2/0 cell was significant nuclear condensation, fragmentation, showing the apoptotic morphology with different SOD concentration after a while, and with the increasing of the concentration, the extent of nuclear condensation and fragmentation increased. SOD can inhibit mouse-borne tumor SP2/0 cell proliferation to some extent in vitro, and showed time-effect and dose-effect relationship, SOD could induce mouse-borne tumor SP2/0 cell apoptosis.
        

猪血中超氧化物歧化酶(SOD)和血红素的提取及SOD的应用研究

摘要9-11
Abstract11-12
第一章 前言13-26
    1 研究的目的和意义13-14
    2 SOD的研究概况14-20
        2.1 SOD的来源、种类及分布14
        2.2 SOD的结构及理化性质14-15
        2.3 SOD是亲水性的蛋白质15
        2.4 SOD的作用底物-自由基15-16
        2.5 自由基的毒性作用16-17
            2.5.1 自由基对脂类和细胞膜的破坏16
            2.5.2 自由基对蛋白质和酶的损害16
            2.5.3 自由基对核酸和染色体的破坏16-17
        2.6 SOD与自由基的作用机制17
        2.7 SOD的应用17-18
            2.7.1 SOD在食品中的应用17
            2.7.2 SOD在临床中的应用17-18
            2.7.3 SOD在化妆品中的应用18
        2.8 SOD与细胞凋亡的关系18-20
            2.8.1 细胞凋亡的形态学特征和生物化学特征19
            2.8.2 细胞凋亡的发生机制19
            2.8.3 细胞凋亡与肿瘤的关系19
            2.8.4 细胞凋亡的意义19-20
            2.8.5 细胞凋亡的检测方法20
    3 血红素的研究概况20-25
        3.1 血红素的结构及性质20-21
        3.2 血红素铁的生理功能21
        3.3 国内外制备血红素的研究进展21-23
            3.3.1 醋酸钠法21-22
            3.3.2 蒸馏法22
            3.3.3 鞣酸法22
            3.3.4 羧甲基纤维素(CMC)法22
            3.3.5 冰醋酸法22
            3.3.6 酶法22-23
            3.3.7 表面活性剂法23
            3.3.8 丙酮法23
        3.4 血红素的应用研究进展23-25
            3.4.1 作为肉制品着色剂23-24
            3.4.2 作为食品的营养强化剂24
            3.4.3 在医学方面的应用24
            3.4.4 在生化试剂方面的应用24-25
    4 本实验主要研究内容及创新点25-26
        4.1 本实验主要研究内容25
        4.2 本实验创新点25-26
第二章 SOD和血红素的提取工艺26-55
    1 材料与方法27-32
        1.1 实验材料27-28
            1.1.1 实验主要试剂27
            1.1.2 实验主要仪器27-28
        1.2 实验方法28-32
            1.2.1 溶血方法的选择28-29
            1.2.2 沉淀血红蛋白方法的选择29
            1.2.3 SOD提取方法的选择29-30
            1.2.4 血红素提取方法的选择30
            1.2.5 SOD分离纯化30
            1.2.6 蛋白标准曲线的制作30-31
            1.2.7 SOD比活力的测定31-32
            1.2.8 血红素标准曲线的制作32
    2 结果与分析32-54
        2.1 蛋白质和血红素的标准曲线32-33
        2.2 以SOD比活力为评价指标的超声波溶血实验33-36
            2.2.1 不同超声功率对SOD比活力的影响33-34
            2.2.2 不同超声时间对SOD比活力的影响34
            2.2.3 不同超声温度对SOD比活力的影响34-35
            2.2.4 正交实验35-36
        2.3 以血红素提取率为评价指标的超声波溶血实验36-39
            2.3.1 不同超声功率对血红素提取率的影响36-37
            2.3.2 不同超声时间对血红素提取率的影响37
            2.3.3 不同超声温度对血红素提取率的影响37-38
            2.3.4 正交实验38-39
        2.4 热变性法提取SOD的实验39-46
            2.4.1 热变性时间对SOD比活力的影响39-40
            2.4.2 热变性温度对SOD比活力的影响40-41
            2.4.3 氯化铜添加量对SOD比活力的影响41
            2.4.4 旋转正交试验结果41-46
        2.5 丙酮沉淀SOD的条件选择46-47
            2.5.1 冷丙酮添加量对SOD比活力的影响46
            2.5.2 冷丙酮沉淀时间对SOD比活力的影响46-47
        2.6 SOD的分离纯化47
        2.7 酸性丙酮法提取血红素的实验47-54
            2.7.1 浸提时间对血红素提取率的影响47-48
            2.7.2 盐酸和丙酮的不同比例对血红素提取率的影响48-49
            2.7.3 酸性丙酮与血红蛋白的不同比对血红素提取率的影响49
            2.7.4 旋转正交设计实验结果49-54
    3 讨论54-55
第三章 SOD和血红素的结构鉴定及稳定性研究55-64
    1 材料与方法55-57
        1.1 实验材料55-56
            1.1.1 实验主要试剂55-56
            1.1.2 实验主要仪器56
        1.2 实验方法56-57
            1.2.1 SOD和血红素的结构鉴定56-57
            1.2.2 SOD和血红素的稳定性研究57
    2 结果与分析57-63
        2.1 SOD和血红素的结构鉴定57-61
            2.1.1 SOD的紫外-可见吸收光谱分析57-58
            2.1.2 血红素的紫外吸收光谱分析58
            2.1.3 血红素的红外吸收光谱分析58-60
            2.1.4 血红素的高效液相色谱分析60-61
        2.2 SOD和血红素的稳定性研究61-63
            2.2.1 SOD的热稳定性实验61
            2.2.2 SOD的储存稳定性实验61-62
            2.2.3 血红素的储存稳定性实验62-63
    3 讨论63-64
第四章 SOD的抗氧化活性及在细胞凋亡中的作用64-76
    1 材料与方法65-69
        1.1 实验材料65-66
            1.1.1 实验主要试剂65
            1.1.2 实验主要仪器65-66
        1.2 实验方法66-69
            1.2.1 SOD测试液66
            1.2.2 SOD的抗氧化活性测定66-67
            1.2.3 SOD在细胞凋亡中的作用67-69
    2 结果与分析69-75
        2.1 SOD抗氧化活性的测定69-71
            2.1.1 还原能力的测定69
            2.1.2 清除羟基自由基的测定69-70
            2.1.3 清除超氧阴离子自由基的测定70-71
            2.1.4 清除DPPH自由基的测定71
        2.2 SOD在细胞凋亡中的作用71-75
            2.2.1 噻唑兰(MTT)比色法测定细胞增殖抑制率71-72
            2.2.2 普通光学显微镜的姬姆萨(Giemsa)染色观察72-74
            2.2.3 荧光显微镜形态学观察(Hoechst 33258)74-75
    3 讨论75-76
第五章 结论76-78
参考文献78-84
致谢84-85
附录85
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