| Phosphates have been used as food additives in meat Processing. Polyphosphate possess versatile functionality:increasing pH and ionic strength, interacting with muscle proteins, promoting dissociation of actomyosin to actin and myosin. Dissociation of the actomyosin complex would also enable the myofibril lattices to expand laterally, thereby allowing an increased water uptake. The increase functionality can allow for increased water retention, improved juiciness and tenderness, enhance palatability. However, polyphosphates added into meat were hydrolyzed irreversibly by the endogenous PPase and TPPase. Pyrophosphatase (PPase) and Tripolyphosphatase (TPPase) was responsible to the hydrolysis of pyrophosphate (PP) and Tripolyphosphate (TPP) in muscle, respectively.In our studies, firstly. PPase and TPPase was purified from pork longissimus dorsi and characterized in detail herein for the first time. Secondly. Determination of the hydrolysis of PP, TPP and polyphosphates blends in PPase or (and) TPPase by free reagent ion chromatography. Finally, the influence of hydrolysis of PP,TPP and polyphosphates blends with time on gel characteristics and rheological characteristics. Following are the results.1 Purification and characterization of pyrophosphatase from pork longissimus dorsiPyrophosphatase (PPase) responsible to the hydrolysis of PP was purified through Ultracentrifugation,50%-70% saturated ammonium sulfate fractionation, DEAE-52 anion-exchange chromatography. The purified enzyme with molecular mass 72 kDa ran as a single band on the SDS-PAGE. A purification fold of 30.7 with activity yield of 8% was obtained in the final step. Enzymes are highly substrate specificity, but in the reaction system, when the concentration of TSPP exceeded 3 mmol·L-1, it would inhibit the PPase activity. The optimum pH and temperature was around 7.5 and 50℃C. respectively. Mg2+ was necessary for PPase and the activity reached a maximum at a concentration of 4.75 mmol·L-1. However. Ca2+ could inhibit PPase. The addition of NaCl and KCl inhibited the enzyme activity and the inhibitory effect of NaCl was stronger than KCl. EDTA-Na2 and EDTA-Na4 activated under low concentrations, but it consumingly did inhibit under high concentrations.1 mmol·L-1NaF completely inhibited the enzyme activity. The kinetic constant Km and Vmax using TSPP as substrate were determined as 0.36 mmol·L-1 and 0.086μmolPi·mg-1 protein·min-1, respectively.2 Purification and characterization of tripolyphosphatase from pork longissimus dorsiMyosin responsible for the hydrolysis of TPP is the major TPPase. Tripolyphosphatase (TPPase) responsible to the hydrolysis of TPP was purified through Ultracentrifugation, 50%-70% saturated ammonium sulfate fractionation. DEAE-52 anion-exchange chromatography. A purification fold of 4.12 with activity yield of 5.65% was obtained in the final step. The optimum temperature and pH was around 40℃and 6.0, respectively. Optimum Mg2+ and Ca2+ concentrations were 0.5 mmol·L-1 and 2.0 mmol·L-1, respectively. PP is a competitive inhibitor of TPPase, and the remaining activity of 7.88% was observed when added at a concentration of 4 mmol·L-1. The TPPase activity decreased as the NaCl and KCl concentration increased and the inhibitory effect of KC1 was stronger than NaCl. EDTA-Na2 and KIO3 inhibited TPPase activity; however Mg2+ is effective in reversal of inhibition caused by EDTA-Na2. The kinetic constant Km and Vmax for the hydrolysis of STPP catalyzed by TPPase were determined as 0.37 mmol·L-1 and 0.049 umolPi·mg-1 protein·min-1. respectively.3 Quantitative analysis of hydrolysis of Polyphosphates in the presence of pure Polyphosphatase enzymeThe aim of our study was to examine the hydrolysis of polyphosphates in the presence of pure Polyphosphatase enzyme by free reagent ion chromatography. PPi was directly to Pi. TPP was firstly hydrolyzed to PP and Pi. and PP was further hydrolyzed to Pi. In the treatments of PPase and TSPP. TSPP was hydrolyzed completely within 50 min. In the presence of TPPase and STPP, only 0.0376g·L-1 of STPP was hydrolyzed in 30 h. the derived PP increased. However, in the coexistence of PPase and TPPase. TPP was completely hydrolyzed in less than 30 h. In the presence of TPPase and STPP and TSPP. only O.O111g·L-1 of STPP was hydrolyzed in 30 h, showing that TSPP inhibited the hydrolysis of STPP. In the treatments of PPase+TPPase and polyphosphate blends. TPP show a considerable decrease in the hydrolysis rate. The hydrolysis of HMP was very slow. The myosin did not hydrolysis PP. PP and TPP in the blends showed a considerable decrease in the hydrolysis rate when the mixture that consisted of PP. TPP and HMP was added in enzyme solution. One of reasons that contributed to the decreased hydrolysis rate of PP and TPP may be the addition of PP possessing an inhibitory effect on TPPase.4 Effect Polyphosphates hydrolysis on rheological properties and gel properties of pork homogenatesTSPP, STPP, polyphosphate blends A (TSPP/STPP/HMP= 2:5:2) and polyphosphate blends B (TSPP/STPP/HMP= 5:2:2) improved proteins solubility and reduced protein turbidity. However, the increase of solubility was not always consistent with the decrease of turbidity. The final storage modulus (G’) values of homogenates gels was increased with polyphosphate added. The polyphosphatase inhibitors delaying hydrolysis of TSPP and STPP, enhanced gel strength and gel retention. With addition of TSPP or STPP alone, there will be a downward trend in water holding capacity (WHC). The downward trend in water holding capacity appeared within 24 h, while the WHC with addition of the phosphates mixture did not fall within 24 h. meat or gel has a better water binding and stability with the addition polyphosphate in mixed form. In our study, the homogenates gel with polyphosphates mixture B added had the best WHC. The addition of polyphosphates little effect on gel elasticity, but could increase the gel strength. The effectiveness of the polyphosphates on gel strength was:polyphosphate blends A> STPP> TSPP> polyphosphate blends B. |
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