【作者】 高瑞昌；

【导师】 薛长湖；

【作者基本信息】 中国海洋大学 ， 食品科学， 2007， 博士

【摘要】 1．建立了水产品中多聚磷酸盐测定的离子色谱法。利用Dionex ICS 2000型离子色谱，AS11-HC型阴离子分离柱，以KOH为淋洗液，采取程序梯度洗脱可以定性定量分析单磷酸盐（Pi）、焦磷酸盐（PP）、三聚磷酸盐（TPP）和六偏磷酸盐（HMP）。0.005-0.5mmol/L的范围内均呈现良好的线性相关性，r²>0.99。在鳙鱼肉中的回收率为99-111%，能够用以测定鳙鱼和其它水产品中多聚磷酸盐。2.对不同水产品中多聚磷酸盐的水解规律进行了研究。离子色谱法可以准确地测定焦磷酸四钠（TSPP）和三聚磷酸钠（STPP）在不同水产中的水解过程。STPP水解分为两步，首先被水解成PP和Pi，其次生成的PP被进一步水解成Pi。而TSPP直接被水解成单磷酸盐。3.利用Dionex ICS 2000型离子色谱，可以非常准确地测定ATP、ADP和Pi。ATP、ADP和Pi在0.01-0.1mmol/L测量浓度范围内具有很好的线性相关性，相关系数均大于0.997，在此基础上建立了腺苷三磷酸酶（ATPase）活性的离子色谱测定法。4.对鳙鱼焦磷酸盐水解酶（PPase）进行了分离纯化和生化特性的研究。肌肉通过0.05 mol/L KCl-20 mmol/L tris-HCl（pH 7.0）缓冲液漂洗得到的粗酶液，经过50％～80％（NH₄）₂SO₄饱和度分段盐析，50℃加热处理，经DE52阴离子交换层析，再经Sephacryl S-300凝胶层析可以得到在native PAGE和SDS-PAGE图谱上呈现单一蛋白条带的纯化酶。纯化倍数为114.5倍，酶活回收率为4.5％。PPase的相对分子量为50kDa，是有两个相同的亚基构成，最适温度和pH分别为50℃和8.0，热稳定性不强，在pH5.0-10.的范围内有着很好的稳定性。Mg²⁺是必需金属离子，当Mg²⁺浓度与底物浓度比值≧1时，酶活性较高且保持稳定；但当比值≦1时，底物对酶有抑制作用。低浓度的EDTA可以激活酶，而高浓度却能强烈抑制酶活。NaBr和二硫苏糖醇（DTT）可以增强酶活性，而柠檬酸钠和亚硫酸氢钠却强烈抑制酶活。PMSF能完全抑制酶活性，该酶可能为丝氨酸酶。在5mmol/LMg²⁺浓度下，PPase水解焦磷酸四钠的Km为1.98mmol/L。5.通过逐层剖析法证明了肌球蛋白头部S-1具有三聚磷酸盐水解酶（TPPase）活性,并阐明了鳙鱼肌球蛋白头部S-1 TPPase的生化特性。纯化的鳙鱼TPPase水解TPP的最适温度为30℃。TPPase在30°C下比较稳定,而在70°C下迅速失活。TPPase最适pH值为5.0和8.0,在中性偏酸的环境中较稳定。Mg²⁺浓度低于17mmol/L时能激活TPPase,但高于17mmol/L却起到了抑制作用。当KCl浓度由0.1 mol/L提高到0.3 mol/L时TPPase活性迅速升高并趋于稳定。EDTA-Na₂能显著抑制TPPase活力。TPPase对于氧化剂和还原剂都比较稳定。在最适条件下测定鳙鱼TPPase的米氏常数为3.2mmol/L。6.研究了三种多聚磷酸盐对肌原纤维蛋白结构的影响,探讨了提高保水性的机理。提出了TSPP提高保水性的机理为:一,通过提高静电斥力使肌原纤维横向膨胀,增加肌原纤维内部的空间;二,从A带两端提取蛋白,增加I带空间;三,从H带提取蛋白,增加A带内部空间;四,焦磷酸盐水解过程中化学键释放的能量可能提供了动力,能破坏肌球蛋白和肌动蛋白的连接,使肌原纤维小片化。STPP作用途径:一方面通过静电斥力使肌原纤维横向膨胀;另一方面提高离子强度增加蛋白质的溶解;第三方面是通过TPPase水解成PPi,然后以PPi形式发挥作用。六偏磷酸盐主要作用可能是通过螯合金属离子影响三聚磷酸盐水解酶和焦磷酸盐水解酶活性,进而影响三聚磷酸盐和焦磷酸盐的作用。7.对磷酸盐替代物进行了研究。利用过氧化氢裂解法获得的分子量大于6kDa的褐藻酸钠裂解物HDX-A能有效的增加南美白对虾虾仁的浸泡增重效果。HDX-A可以显著保持冷冻虾仁浸泡增重效果,防止了解冻损失,降低了蒸煮损失。在南美白对虾冷冻虾仁应用效果优于B542复合磷酸盐。提出HDX-A提高冷冻虾仁的保水性的可能机理为:一,通过渗透作用进入到虾仁内部,利用自身膨胀增加了肌束之间和肌原纤维之间的空间,并形成空间上的网络结构,增强了结构的稳定性;二,与肌原纤维蛋白结合,增强蛋白质的稳定性,防止肌球蛋白的变性;三,大分子量的裂解物可以吸附并保持大量的水分子;四,在虾仁表面通过Ca²⁺、Mg²⁺等离子形成凝胶膜,阻止了内部水分的流失。更多还原

【Abstract】 1. An ion chromatography method was established for determine orthophosphate （Pi）, pyrophosphate （PPi）, tripolyphosphate （TPP）, and hexametaphosphte （HMP）. The method was linear at concentration raging from 0.005-0.5mmol/L. The average recoveries ranged form 99-111%. The method can be applied for determination of phosphates in aquatic products.2. The hydrolysis of PPi and TPP in several kinds of aquatic products was determined. TPP was firstly hydrolyzed to PPi and Pi, and PPi was further hydrolyzed to Pi in minced meat. Meantime, PPi was directly to Pi.3. An innovative ion chromatography method was established for determine Pi, adenosine diphosphate （ADP）, and adenosine triphosphate （ATP）. The method can be applied for determination of ATPase activity. 4. Pyrophosphatase （PPase） responsible to the hydrolysis of PPi was purified from bighead carp （Aristichthys nobilis）, and characterized in detail herein. PPase was extracted with 0.05mol/L KCl buffer （pH 7.0）, followed by heat treatment and ammonium sulfate precipitation. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography. The purified PPase was obtained. The molecular mass was 50 kDa with two subunits. The optimum pH and temperature was around 8.0 and 50°C, respectively. Mg²⁺ was necessary. An excess of PPi over Mg²⁺ resulted in inhibition of PPase. EDTA activated under low concentrations, but it consumingly did inhibit under high concentrations. NaBr and DTT could enhance PPase activity. PMSF, Na₃C₆H₅O₇ and NaHSO₃ could significantly inhibit PPase. PPase converted PPi to Pi stoichiometrically with a K_m of 1.98 mmol/L.5. The myosin subfragment-1 was proved to have the activity of tripolyphosphatase （TPPase）. The optimum temperature and pH for the TPPase activity were 30°C and pH 5.0 and 8.0. Mg²⁺ was necessary and the optimum concentration was 17 mmol/L. The optimum concentration of KCl for the TPPase was 0.3 mol/L. The TPPase was significantly inhibited by EDTA-Na₂. The TPPase converted TPP to PPi and Pi stoichiometrically with a K_M of 3.2 mmol/L.6. The effects of polyphosphates on the microstructure of myofibrillar were studied. The mechanism of PPi on water holding capacity was described as: （i） enabled the myofibrilar lattices expand laterally; （ii） increased the space of I- band by extracting protein from both ends of A-band; （iii） increased the space of A-band by extracting protein from H-band; （iv） destroyed the juncture of the myosin and actin by the energy liberated from the hydrolysis of chemical bond. The mechanism of TPP was described as: （i） enabled the myofibrilar lattices expand laterally; （ii） increased the soluble protein content by the ion strength; （iii） firstly hydrolyzed to PPi, which effectively destroyed the structure of myofibrillar. HMP could chelate metal ion that affected the hydrolysis of PPi and TPP. As a result, the function of PPi and TPP was modified.7. A safe effective phosphate alternative was exploited. The product of molecular weight over 6000 from sodium alginate obtain by H₂O₂ significantly increased the weight of white leg shrimp, and decreased the frozen drip loss. The effect of HDX-A preceded polyphosphate compound. The mechanism of HDX-A was presumed as follows: （i） penetrated into shrimp, swelled, and expanded the space of myofibrils or fascicles; （ii） combined with protein, prevented myosin frozen denaturation; （iii） adsorbed and retained entrapped water; （iv） formed a film and prevented frozen drip loss.更多还原