【作者】 汪学荣；

【导师】 阚建全； 邓尚贵；

【作者基本信息】 西南大学 ， 农产品加工及贮藏工程， 2008， 博士

【摘要】 缺铁性贫血是体内贮存铁不足,影响血红蛋白合成所引起的一种细胞低色素性贫血,是世界各地贫血中最常见的一种。据WHO报道,全世界约有10%～30%的人群有不同程度的缺铁,男性发病率约10%,女性大于20%。目前,缺铁性贫血的防治主要通过摄入补铁剂来实现。常见的补铁剂有硫酸亚铁、氯化亚铁、葡萄糖酸亚铁、乳酸亚铁、琥珀酸亚铁和富马酸亚铁等,这些补铁剂虽然铁含量高,补铁效果较好,但是由于它们在体内的利用率较低,毒副作用大,并且有特殊的金属铁锈味。多肽螯合铁是蛋白质水解生成的多肽与二价铁离子螯合而生成的生物态铁,在体内不受碳酸盐、单宁酸和纤维素等干扰物质的影响,在体内始终处于可吸收的二价状态,可以直接被肠粘膜细胞吸收,无毒副作用,不产生任何消化道刺激症状,生物利用率高,是一种理想的补铁剂。我国是世界上生猪出栏数最多的国家,年出栏生猪4～5亿头,每头猪约有2kg猪血,因此每年由屠宰生猪而产生的副产物猪血大约有8～10亿kg,这相当于400万吨猪肉或460万吨全蛋所含的蛋白质。猪血虽然营养丰富,但因其色泽差,血腥味重,适口性差,血红蛋白不易消化吸收,因而未被很好利用。目前,我国的猪血除部分作食用和加工血粉外,还有约70%的猪血被废弃掉,不但浪费了宝贵的资源,而且污染了环境。因此,本论文以猪血粉为原料,使用碱性蛋白酶进行水解,再将膜分离纯化得到的猪血多肽与亚铁盐进行螯合反应,然后对制得的猪血多肽铁螯合盐进行理化性质研究、功能性质研究和食品毒理学安全性评价,以期为缺铁性贫血人群提供理想的补铁制剂,并为其推广和应用提供理论依据和有益参考。本论文主要获得了以下研究结果:1、猪血酶法水解制备猪血多肽的研究1.1 2709碱性蛋白酶对猪血粉具有较好的水解效果,其水解最佳工艺参数为:pH值10.5,水解温度45℃,底物浓度2%,酶与底物质量比3.0%,水解时间6h。1.2活性炭对猪血粉酶解液具有理想的脱色效果,其最佳脱色工艺参数为活性炭用量2.5%,pH4.0,脱色温度60℃,吸附时间1.0h,在此最佳脱色工艺条件下猪血粉酶解液的脱色率达92.20%,同时氮损失率为10.42%。1.3离子交换树脂对猪血粉酶解脱色液具有较好的除盐效果。当猪血粉酶解脱色液进样速度为2BV/h时,除盐率达82.11%,同时氮损失率为6.59%。聚丙烯中空纤维超滤膜对除盐后的猪血粉酶解多肽液具有很好的截留分离效果,超滤的最佳操作压力为0.15MPa,多肽液最适温度为25℃,在最佳操作压力和最适温度条件下得到了10kDal以上、5～10kDal、1～5kDal和1kDal以下4个相对分子质量段的猪血多肽,多肽得率分别为4.38%,13.97%,74.76%和6.89%。可见,猪血多肽相对分子质量主要集中于1～5kDal之间。1.4制备得到的猪血多肽呈浅黄色粉末状,无血腥气味,口感较细腻,味道适中,略带苦味。2、猪血多肽铁螯合盐的制备及成分分析2.1从硫酸亚铁和氯化亚铁在不同浓度乙醇中的溶解性可知,氯化亚铁能完全溶于不同浓度的乙醇溶液中,更适合作为螯合反应的铁源。2.2猪血多肽铁螯合盐制备的最佳工艺参数为:pH值5,多肽与氯化亚铁质量比4:1,多肽溶液浓度3.0%。2.3通过硫化钠法定性检测螯合反应产物可知,猪血多肽铁是以多肽螯合物形式存在的,是不同于猪血多肽和氯化亚铁的一种新物质,并且其主要成分是多肽和铁,二者质量百分数分别为70.02%和12.23%。3、猪血多肽铁螯合盐的理化性质研究3.1猪血多肽铁螯合盐能溶于水中,在中性条件下其溶解度最小,随着酸性或碱性的增强,溶解度增加;猪血多肽铁螯合盐难溶于有机溶剂。3.2猪血多肽铁螯合盐的外观、固体和溶液的稳定性均优于硫酸亚铁。3.3经过初步的紫外扫描分析和红外光谱分析可知,猪血多肽铁螯合盐与猪血多肽、氯化亚铁不同,是一种新型螯合物。4、猪血多肽铁螯合盐的抗氧化和抗贫血功能研究4.1猪血多肽铁螯合盐能清除超氧阴离子自由基（O₂^-·）和过氧化氢（H₂O₂）,维生素C、猪血多肽铁和猪血多肽这三种物质对超氧阴离子自由基（O₂^-·）和过氧化氢（H₂O₂）清除能力的强弱顺序为维生素C＞猪血多肽铁＞猪血多肽,因此猪血多肽铁螯合盐具有较强的清除超氧阴离子自由基（O₂^-·）和过氧化氢（H₂O₂）的能力。4.2通过低铁饲料喂养,辅以每周尾静脉放血的方法,4周后可以成功建立大鼠缺铁性贫血模型。用猪血多肽铁螯合盐灌胃缺铁性贫血大鼠4周后可使缺铁性贫血大鼠的血红蛋白、红细胞计数、血清铁水平显著升高（P＜0.05）,故猪血多肽铁螯合盐可以明显改善大鼠缺铁性贫血,并且其抗贫血效果明显优于葡萄糖酸亚铁和氯化亚铁。5、猪血多肽铁螯合盐的安全性评价5.1经口急性毒性(LD₅₀)试验表明,小鼠经口灌喂猪血多肽铁螯合盐后无毒性反应,由寇氏改良法计算公式得到猪血多肽铁螯合盐的LD₅₀=30 998mg/kg,大于15000mg/kg,故猪血多肽铁螯合盐为实际无毒物质。5.2 Ames试验表明,猪血多肽铁螯合盐的5个剂量组在加S-9或不加S-9活化的情况下,4个菌株TA97、TA98、TA100和TA102的回变菌落数与阴性对照组较接近,并且没有超过阴性对照组的2倍,而与阳性对照组相比要低很多,故猪血多肽铁螯合盐不能使鼠伤寒沙门氏菌移码突变株和碱基置换突变株发生回复突变的现象,未呈现致突变性。微核试验和畸形试验表明,阳性对照组（环磷酰胺组）细胞微核率和精子畸形率与阴性对照组相比较,差异具有极显著性（p＜0.01）,而猪血多肽铁螯合盐各剂量组细胞微核率和精子畸形率与阴性对照组相比较,差异均不显著（p＞0.05）,并且无剂量-效应关系。因此,猪血多肽铁螯合盐对小鼠骨髓细胞染色体无断裂效应,对小鼠精子的畸形无影响。5.3大鼠30天喂养试验表明,三个剂量试验组的大鼠生长发育与行为活动正常,动物体重和食物利用率与阴性对照组比较均无显著性差异（P＞0.05）;各剂量组大鼠血液生化指标与阴性对照组比较均无显著性差异（p＞0.05）,且无剂量-效应关系;各剂量组的脏体比与阴性对照组相比较均无显著性差异（p＞0.05）,器官组织病理学检查未见异常。更多还原

【Abstract】 Iron deficiency anemia（IDA）,one of the most common anemia in the world,is a kind of cell hypochromic anemia due to insufficient iron which is needed to make hemoglobin.According to WHO,about 10%to 30%of the whole world population are suffered from different degrees of iron deficiency,the incidence of men by about 10%, 20%more than women.At present,prevention and treatment of iron deficiency anemia is mainly achieved through the intake of iron agents such as ferrous sulfate,ferrous chloride,ferrous gluconate,ferrous lactic,ferrous succinate and ferrous fumarate.These agents have good effects in iron supplementation for their high iron content,whereas they can be relatively low utilized in the body,and have side effects and a special taste of metal rust.Polypeptide chelated iron,a biostate iron chelated and generated by polypeptide from albuminolysis and ferrous iron ion,is an ideal agent for it can not be affected by such interferents as carbonate,tannic acid and cellulose,but can always be absorbed directly by intestinal cells without any toxic side effects and irritation.China is one of the biggest pig producing countries,slaughtering 4 - 500,000,000, pigs each year.And each pig has about 2kg blood,so about 8 to 1,000,000,000 kg pig blood will be produced each year,which is equivalent to the protein content of 4,000,000 tons of pork or 4,600,000 tons of eggs.While the pig blood is nutritious,but because of its poor color,heavy blood smell,poor palatability and non-digestible absorption of hemoglobin,it has not been fully utilised.At present,only less than one third of the pig blood in China is processed as food and blood powder,and there are about 70%of it abandoned.That leads to a waste of valuable resources and environmental pollution.So,in this paper the pig blood powder has been used to be raw materials,and been hydrolysed by alkaline proteinase,and then the chelating reaction has been conducted between the polypeptides separated and purified by film from the pig blood and ferrite. And then physicochemical properties and functional properties of the chelated pig blood polypeptide-iron salt have been studied,and its toxicological safety has been evaluated with a view to providing a ideal supplementation iron agent for people who are iron deficiency anemia.Finally,the theoretical basis for its promotion and application and a useful reference have been provided.This paper consists of the following findings:1、Research on preparation of pig blood polypeptides by means of enzymatic hydrolysis1.1 Concerning hydrolysis,2709 alkaline protease had relatively good effects on pig blood powder,and the optimum hydrolysis technology parameters were:pH value,10.5; hydrolysis temperature,45℃;concentration of substrate,2%;the ratio of enzyme and the substrate quality,3.0%;hydrolysis time,6h..1.2 Activated carbon had good decolorization effects on the enzymolysed liquid of pig blood powder,and the best decolorization process parameters were:the dosage of activated carbon,2.5%;pH value,4.0;decolorization temperature,60℃;adsorption time, 1.0h.Under the best decolorization conditions the decolorization rate of the enzymolysed liquid of pig blood powder was 92.20%,while the loss rate of nitrogen was 10.42%.1.3 Ion exchange resins had relatively good demineralization effects on the decolorized enzymolysis liquid of pig blood powder.The demineralization rate was 82.11%when the sample injection speed of decolorized enzymolysis liquid of pig blood powder was 2BV/h,while the loss rate of nitrogen was 6.59%.Polypropylene hollow fiber ultrafiltration membrane had relatively good effects of retention and separation on the demineralized enzymolysis polypeptides liquid of pig blood powder.The optimum operation pressure of ultrafiltration was 0.15MPa,and the optimum temperature of polypeptides solution was 25℃.Under the conditions of optimum operation pressure and optimum temperature the pig blood polypeptides of four relative molecular mass segments were obtained,and they were the segments above 10kDal,5～10kDal,1～5kDal and the segment below 1kDal.The rates of polypeptide yield were 4.38%,13.97 %,74.76%and 6.89%respectively,and the relative molecular mass concentrated on 1～5kDal.1.4 The yielded pig blood polypeptides are in the form of light yellow powder with delicate mouth.feel and taste,slightly bitter but no smell of blood.2、Preparation and component analysis of the chelated salt of pig blood polypeptide-iron2.1 From dissolubility of ferrous sulfate and ferrous chloride in different concentrations of ethanol,we could see that ferrous chloride could be completely dissolved in different concentrations of ethanol solution,and it was more suitable for it to be used as the iron source in the chelation.2.2 The optimum technology parameters of the preparation of the chelated salt of pig blood polypeptide-iron were:pH value,5;the mass ratio of polypeptides and ferrous chloride is 4:1;the polypeptides solution concentration 3.0%.2.3 From the qualitation measurement of the chelating reaction products by sodium sulfide method we could see that the pig blood polypeptides iron existed in the form of polypeptides chelate compound,and it was a new material,and it was different from the pig blood polypeptides and ferrous chloride,and its major components were polypeptide and iron,the mass percentages of the two were 70.02%and 12.23% respectively.3、Study on the physicochemical properties of the chelated salt of pig blood polypeptide-iron3.1 The chelated salt of pig blood polypeptide-iron could be soluble in water,and under the neutral conditions,it had the minimum solubility,and with the increase of acidity or alkalinity,the solubility rised;and the chelated salt of pig blood polypeptide-iron could not be dissolved in organic solvents.3.2 The chelated salt of pig blood polypeptide-iron were more stable in appearance and the state of solid and liquid than ferrous sulfate.3.3 Through a preliminary scan of the ultraviolet and infrared spectroscopy analysis,we could see that the chelated salt of pig blood polypeptide-iron was different from pig blood polypeptide and ferrous chloride,and it was a new type of chelate.4、The study on the antioxidant and anti-anemia function of the chelated salt of pig blood polypeptide-iron4.1 The chelated salt of pig blood polypeptide-iron could remove superoxide radical anion（O₂.） and hydrogen peroxide（H₂O₂）.The remove ability of the chelated salt of pig blood polypeptide-iron was weaker than that of vitamin C,but stronger than that of pig blood polypeptides,so the chelated salt of pig blood polypeptide-iron had relatively strong ability of removing superoxide radical anion（O₂） and hydrogen peroxide （H₂O₂）.4.2 To establish the model of rat iron deficiency anemia successfully,it involved bloodletting caudal vein each week and feeding it with low iron feed for four weeks. The rat’s hemoglobin,the red blood cell count and serum iron levels increased significantly（P＜0.05）,fed with the chelated salt of pig blood polypeptide-iron after 4 weeks.It was obvious that the chelated salt of pig blood polypeptide-iron could improve the rat’s iron deficiency anemia,and its anti-anemia function was better than that of ferrous gluconate and ferrous chloride.5、Safety evaluation of the chelated salt of pig blood polypeptide-iron5.1 Acute oral toxicity(LD₅₀) test showed that the mice had no toxic reaction after being fed with the chelated salt of pig blood polypeptide-iron by mouth.Counted by the improved formula of Karber method the LD₅₀ of the chelated salt of pig blood polypeptide-iron was 30 998 mg / kg,and it was more than 15 000mg/kg,so it could be concluded that the chelated salt of pig blood polypeptide-iron was innocuous substance. 5.2 Ames test showed that under the conditions of activating the five groups of different dose by adding S-9 or not,the number of anaplasia bacterial colony of the 4 strains, TA97,TA98,TA100 and TA102,was close to that of the negative control group,and less than 2 times.And compared with positive control group the number was much lower.So it could be concluded that the chelated salt of pig blood polypeptide-iron could not lead to back mutation between the Salmonella typhimurium mutation strain and base replacement mutant,and it had no mutagenicity.Micronucleus and abnormality test showed that the difference in the rate of cell micronuclear and abnormal sperm between the positive control group（fed with cyclophosphamide）and the negative control group was significant（p＜0.01）,and that between the negative control group and the groups fed with different dose of the chelated salt of pig blood polypeptide-iron was not significant（p＞0.05）,and there’s no dose - effect relationship between them.So the chelated salt of pig blood polypeptide-iron had no abruption effect on mice bone marrow cells chromosome,and had no effect on mice abnormal sperm.5.3 The test of feeding the mice with the chelated salt of pig blood polypeptide-iron for 30 days showed that the growth development and behaviour of mice of the three groups of different dose were normal,and compared with that of the negative control group the differences in body weight and food utilization were not significant（p＞0.05）.Compared with that of the negative control group,the differences in blood biochemical indicator were not significantly different（P＞0.05）,and there’s no dose-effect relationship between them.Compared with that of the negative control group,the differences in the ratio of viscus and corpora were not significant（p＞0.05）,and there is no abnormality in the test of organ histopathology.更多还原