【作者】 朱媛媛；

【导师】 庄红；

【作者基本信息】 吉林大学 ， 食品科学， 2011， 硕士

【摘要】 亚铁血红素不仅具有抗贫血作用,而且具有抗氧化清除自由基的功能,本研究主要对亚铁血红素的制备、保护、纯化及其功能性进行研究。采用亚硫酸钠还原和吡啶-氢氧化钠显色法绘制亚铁血红素的标准曲线。曲线精密度偏差为0.449%,重现性0.4813%,显色稳定,回收率置于置信区间;高铁血红素的标准曲线R~2为0.9999,回收率置于置信区间;高效液相色谱法绘制标准曲线,精密度RSD为0.70%,重现性2.02%,回收率置于置信区间。表明标准曲线准确度很高,方法可行。复合酶解牛血红蛋白制取亚铁血红素。对两种酶底物浓度、酶浓度、pH值和酶解时间进行单因素、正交和回归实验。且采用单一抗氧化剂、还原剂及抗氧化剂/还原剂组合保护亚铁血红素中的二价铁。结果表明:碱性蛋白酶底物浓度7%、加酶量1%、pH值8、酶解2h,风味酶pH值为6.5、加酶量2%、酶解2h,可得到最好水解效果,此时,亚铁血红素得率11.18mg/ml;保护剂L-抗坏血酸棕榈酸酯(0.03%)/亚硫酸钠(0.03%)的亚铁保护性能最强,亚铁血红素得率可达12.60mg/ml,与无保护剂添加比提高了11.27%;酶解产物露置168h后亚铁血红素得率可达到7.44 mg/ml,与未添加比提高了6.85%。采用酸碱法和盐析法进行亚铁血红素的纯化,酸碱法纯化单因素为碱浓、料液比和pH值。进行了RSM模型,获得最优条件为碱浓0.5%、料液比1:15、pH为6,血红素铁纯度9.63%,得率3.48mg/ml;盐析法纯化单因素为碱浓、料液比、钙盐添加量。获得最优纯化条件为:碱浓1%、料液比1:10、钙盐添加量1g,得率为2.71mg/mL,纯度为60.91%。实验发现,盐析法优于酸碱纯化法。对制得的亚铁血红素进行了抗氧化实验。体外抗氧化实验主要进行了DPPH自由基、过氧化氢自由基、还原力能力和总抗氧化能力测定。亚铁血红素的DPPH自由基清除率随浓度增加而上升,为63.24%-65.75%;过氧化氢自由基清除率随浓度的增加而增强,清除率为抗坏血酸的15%-85%;还原力与浓度呈正相关,为抗坏血酸的12%-69%;总抗氧化能力随浓度增加而增高,约为抗坏血酸的5%-11%。体内抗氧化性研究中,亚铁血红素治疗组可明显提高CAT含量、SOD活力、GSH水平和降低MDA含量,而且中剂量组T-AOC的均值最高,表明中剂量组亚铁血红素组具有最高的抗氧化活性。对制得的亚铁血红素进行了铁吸收效果实验。与对照组相比,大鼠对亚铁血红素的生物利用率很高。分别进行了体重、脏器系数、血红蛋白浓度、红细胞数量、红细胞压积、平均血红蛋白浓度、白细胞数量、中性粒细胞数量、中性粒细胞比率和血清铁指标的影响研究。实验发现,中剂量亚铁血红素的供给组的各项指标显著高于对照组,表明中剂量更适宜作为大鼠的补充剂量。更多还原

【Abstract】 Heme iron processes anti-anemia, free radical scavenging and anti-oxidant functions.We mainly studied its preparation, protection, purification and functionality.We used sodium sulfite reduction and pyridine sodium hydroxide chromogenic method to draw a standard curve. The cure precision RSD was 0.449%, the reproducibility RSD was 0.4813%, colouration stable, and the recovery placed in confidence interval. R² of hemin standard curve was 0.9999, the recovery also placed in confidence interval. HPLC method was used to draw a standard curve, the precision RSD was 0.70%, the reproducibility RSD was 2.02%, and the recovery placed in confidence interval. The results indicated that the standard cure was precise, the method was feasible.We used compound enzyme for hemoglobin enzymolysis. Single factor, orthogonal and regression experiments were selected to optimize hydrolysis conditions which affected the concentration of substrate, enzyme concentration, pH and enzymolysis time of the two enzymes. Single antioxidants, single reductant, composite antioxidants and antioxidant/reductant were added to protect ferrous iron. The optimum conditions were: substrate concentration 7%, Alkaline concentration 1%, pH 8, hydrolysis time 2h; flavor enzyme concentration 2%, pH 6.5, hydrolysis time 2h; the yield of heme achieved 11.18mg/ml. Protective agents anhydrous sodium sulfite（0.03%）/l-ascorbyl palmitate （0.03%） proved to be the optimum for ferrous iron protection, ferroprotoporphyrin yield reached 12.60mg/ml, which raised 11.27%.The enzymatic hydrolysate was exposed 168h then, the yield was 7.44mg/ml, which raised 6.85%.Acid-alkali method and salting-out method was used to purify heme iron. The single factors of acid-alkali method were alkali concentration, solid-liquid ratio, and pH value. We designed the RSM model to obtain the optimal conditions: alkali concentration 0.5%, solid-liquid ratio 1:15, pH value 6, purity 9.63%, yield 3.48mg/ml. The single factors of salting-out method were alkali concentration, solid-liquid ratio, and calcium salt addition. The optimal conditions were: alkali concentration 1%, solid-liquid ratio 1:10, calcium salt addition 1g, purity 60.91%, yield 2.71mg/ml. It was evidenced that salting-out method proved to be better than acid-alkali method.We carried out antioxidation experiment of heme iron. In the in vitro assays, we mainly determined scavenging of radical DPPH anions, H₂O₂ free radical, reduce power and total antioxidant activity. The scavenging rate of DPPH radical was 63.25-65.75% with the rise of heme iron concentration. The scavenging rate of H₂O₂ free radical was 15-85% of ascorbic acid with the rise of heme iron concentration. The reduce power was 12-69% of ascorbic acid with the rise of heme iron concentration. The total antioxidant activity was 5-11% of ascorbic acid with the rise of heme iron concentration. Heme iron also showed strong suppressive effect in vivo assays. The results indicated that intragastric administration of heme iron could increase levels of CAT, SOD, T-AOC, GSH and decrease MDA content. The middle-heme group showed highest T-AOC mean. It indicated that the middle-heme group possessed the highest antioxidant activity.Heme iron absorption effect experiments were also designed in the iron-deficient rats showed high bioavailability of heme iron, compared with the positive control group. We determined body weights, organ coefficient, hemoglobin concentration, RBC hematocrit, average hemoglobin concentration, leukocyte, neutrophil, neutrophile ratio and serum iron. The results showed that each index of middle dose group of heme was higher than control group, middle dose of heme iron improved iron supplement significantly.更多还原