Hydrolytic and enzymatic breakdown of food grade condensed phosphates in white shrimp (Penaeus setiferus)

Abstract

Ortho- and condensed phosphates were separated and identified on cellulose coated plates using an improved thin layer chromatographic technique. In addition to qualitative identification, this method is also well suited for quantitative determination of polyphosphate mixtures. The lowest level of detection for both ortho- and condensed phosphates after TLC separation was 0.08% on a weight basis. The average recoveries of ortho-, pyro-, and tripolyphosphate from shrimp muscle after TLC separation were found to be 90%, 96% and 97%, respectively. By using the TLC procedure, polyphosphates in shrimp stored at -25°C were shown to be stable over a 10 month time interval. However, when STPP and SHMP were incubated with shrimp extracts at 5, 10, 25, and 35°C, there was a rapid breakdown of the polyphosphates to orthophosphates. Since the rate of hydrolysis in cooked extracts was much slower, endogenous phosphatase enzymes were capable of catalyzing the hydrolysis of polyphosphates in treated shrimp. The activation energy of the shrimp phosphatase-catalyzed hydrolysis of sodium tripolyphosphate, as calculated from an Arrhenius plot, was 35 Kcal/mole. Phosphatases were semi-purified from shrimp extracts by ammonium sulfate precipitation and gel filtration. According to elution volumes from a Sephadex G-200 column, the molecular weight of shrimp phosphatase was calculated to be approximately 200,000. However, after SDS-gel electrophoresis, the active phosphatase fraction migrated according to a molecular weight of 90,000. This indicates that the enzyme may consist of two active subunits not separated through gel filtration. The optimum pH of the enzyme was 8.5 with maximum activity at 35°C assay temperature. The enzyme is stable at low temperatures but loses its activity rapidly when stored at 35°C.