Study On The Mechanism Of Polyphosphates Hydrolysis In Bighead Carp And Non-phosphate Additive

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.