【作者】 杨娟；

【导师】 李志良；

【作者基本信息】 重庆大学 ， 分析化学， 2008， 硕士

【摘要】 猪血是一种非常宝贵的生物资源,可以利用猪血提取多种生化产品,如氯化血红素、凝血酶、白蛋白、超氧化物歧化酶(SOD)等产品,蕴藏着巨大的潜力和利用价值。其中氯化血红素除了可用作食品中的色素添加剂,还是目前治疗缺铁性贫血疗效较好的一种补铁剂,具有生物利用率高、无体内铁蓄积中毒及胃肠刺激等不良反应的优点,同时还是抗贫血和抗肿瘤药物的重要原材料。从动物血液中提取得到的血红素产品一般是氯化高铁血红素,本文分别研究了冰醋酸法、酸性丙酮法、CMC-Na法制备氯化血红素,对产品的产量、纯度、收率进行了分析,并对产品进行了定性鉴定。最后对三种制备方法进行了横向、系统的对比研究,为制备氯化血红素的工业生产提供了理论依据。采集新鲜猪血,对猪血进行抗凝处理,分离红细胞和血浆。用3.8%的柠檬酸钠溶液作为抗凝剂,抗凝时柠檬酸钠溶液与血液按体积比1:9混合。抗凝血在3000 -4000r/min的转速下离心分离,离心分离10min,收集红细胞,用于制备氯化血红素。取30mL红细胞,用冰醋酸法制备氯化血红素,在冰醋酸法中,探讨了冰醋酸添加量、反应温度、反应时间、氯化钠添加量对制备氯化血红素的影响,通过分析对比氯化血红素标准品和样品的性状和紫外/可见光谱图,确定所得产品为氯化血红素,经实验结果分析,得出最佳制备条件是冰醋酸添加量为血球的4.5倍,反应温度确定为105℃,反应时间为35min,氯化钠的添加量为血球的1.5%时制备得到的氯化血红素收率最高。按所得到的最佳工艺条件进行三次平行实验,氯化血红素的收率和纯度平均可达到38.12%和26.35%。对酸性丙酮法制备氯化血红素的工艺进行了研究,通过单因子实验和正交实验分别研究了加水量、超声波破碎时间、酸性丙酮加入量、pH值、乙酸钠加入量对制备氯化血红素的影响,结果表明制备氯化血红素的最佳条件是酸性丙酮加入量为150mL,pH值确定为4.8,乙酸钠加入量为滤液体积的2%时制备得到的氯化血红素收率最高。在此条件下进行三次平行实验,氯化血红素的收率和纯度平均可达到44.88%和36.30%。本文在前人实验的和预实验的基础上,确定了CMC-Na法中沉淀珠蛋白溶液pH值为3.2,搅拌时间为30min,CMC-Na悬浮液浓度为1.2%,提取搅拌时间为50min,考查了CMC-Na加入量、吸附时间、pH值三个因素对制备氯化血红素的影响,实验结果表明,最佳工艺条件是CMC-Na加入量为0.45g,吸附时间为8h,pH为6.0时制备得到的氯化血红素收率最高。按所得到的最佳工艺条件进行三次平行实验,所得氯化血红素的收率和纯度平均值分别为56.05%和29.42%。由CMC-Na法制备所得氯化血红素产品不需要洗脱,性质稳定,可以久存,并且所得产品可以直接制成口服液或压缩片,不影响其生物学效应,完全可以达到补铁的效果,该法分离出来的血红蛋白无有机溶剂污染可再利用。分别在对三种方法的工艺进行了系统研究的基础上,首先在感官下进行了对比,以产量、纯度和收率为评价指标,比较了三种方法制备氯化血红素的效果,几种方法制备得到的产物纯度大小依次是:酸性丙酮法〉CMC-Na法〉冰醋酸法,收率大小依次是:CMC-Na法〉酸性丙酮法〉冰醋酸法。但纯度可以通过经过重结晶提高,所以总的来说,那种方法所得氯化血红素收率越高,这种方法也就越好,所以在三种方法种,CMC-Na法制备氯化血红素的效果最好,其收率可以达到56.05%。更多还原

【Abstract】 Swinish blood is a kind of valuable biological resources, variety biochemical products can be extracted from swinish blood, such as hemin, thrombin, albumin, superoxide dismutase (SOD), and other products. It contains enormous potential and value. Hemin which can be used as the pigment additives in food, and can be used as a better efficacy of iron supplementation with high bioavailability to treat the current iron deficiency anemia ,it will not cause adverse reactions of poisoning because of iron accumulation ,At the same time, it is a kind of important raw materials which can be used as anti-anaemia and antineoplastic agents.The products which prepared form swinish blood always are hemin,not heme.The paper studied hemin which prepared by glacial acetic acid, acetone acid and CMC-Na. Product yield, purity and yield were analyzed,the products were identified qualitatively. Finally, three methods of hemin preparation were horizontal, systematic comparative studied.It provides a theoretical basis for hemin preparation of industrial production.Collecting fresh swinish blood, doing anticoagulant treatment ,he separating red blood cells and plasma from swinish blood . 0.38% Sodium citrate solution was added into the fresh swinish blood according to the volume proportion of 1:9.The anticoagulant blood was centrifuged for 10 min at 3000-4000 r/min to collect RBC for preparation of hemin.When prepare hemin by acetic acid,we got 30 mL red blood cells.In the acetic acid method, the amount of acetic acid, the reaction temperature, reaction time and sodium chloride content on the impact of hemin were studied, the production was con- finned to be hemin by respectively analyze the infrared spectrogram and ultraviolet /visible spectrogram of hemin sample and hemin standard, The optimum conditions for the extraction techniques of hemin were: separating hemin from erythrocyte at 4.5 acetic acid supplement,at 105℃reaction temperature,at 35min reaction time, at 1.5% Sodium Chloride supplement,Doing three parallel experiments according to the optimum conditions, the average yield and purity of hemin can be achieved 38.12% and 26.35%.Preparation of hemin by acidic acetone were studied, the impact of hemin Pre- paration by the water content, ultrasonic broken time, the amount of acid acetone, pH value, the amount of sodium acetate were studied through Single-factor and ortho- gonal experiment. Preparation of hemin results show that the best conditions were: add 150mL acidic acetone, pH value was 4.8,add 2% sodium acetate.Doing three parallel experiments according to the optimum conditions, the average yield and purity of hemin can be achieved 44.88% and 36.30%.the paper Based on previous experiments and pre-experiment, pH value of 3.2 were identified in the Act globin protein precipitation solution by CMC-Na, mixing for 30 min, CMC-Na suspension concentration of 1.2%, the stirring time for 50 min, CMC-Na addition, the adsorption time, the pH value on the impact of preparation of hemin were studied. Preparation of hemin results show that the best conditions were:add 0.45g CMC-Na, adsorption time was 8h, pH value was 6.0, Doing three parallel experiments according to the optimum conditions, the average yield and purity of hemin can be achieved 56.05% and 29.42%. hemin products prepared by CMC-Na do not need clear, they are stable, can be made directly to oral liquid or compressed to tablets, and does not affect its biological effects,and can be achieved the effect of iron supplementation, Hemoglobin seperated by this method can be recycled as it has not pollution with organic solvent.Under the study of three methods, first of all,the products were compared by eyes,then product quality, yield and purity of hemin were compared by three methods to find the best method. The purity of the product were: acidic acetone method> law CMC-Na> acetic acid. The yield of the product were: CMC-Na Act> acidic acetone method> acetic acid method.But purity can be improved by recrystallization, in general, the higher yield of hemin was, the better this way was,so among three method, CMC-Na is the best way to prepare hemin ,its yield can be achieved 56.05%.更多还原