Identification by GeLC‐MS/MS of Trypsin Inhibitor in Sarcoplasmic Proteins of Three Tropical Fish and Characterization of Their Inhibitory Properties

Sarcoplasmic proteins from 3 fish species were fractionated by 50% to 70% ammonium sulfate precipitation. Lyophilized fractionated sarcoplasmic proteins of threadfin bream (TB‐SP), bigeye snapper (BS‐SP), and yellow croaker (YC‐SP) showed 80% to 92% trypsin inhibitory activity. Trypsin inhibitory activity staining gel electrophoresis revealed bands at 32, 33, 37, 45, 48, and 50 kDa for the 3 species, and a band at 95 kDa was observed for TB‐SP and YC‐SP. Alpha‐1‐antitrypsin with molecular mass of 45 to 50 kDa was identified in YC‐SP by gel‐based liquid chromatography‐tandem mass spectrometry (GeLC‐MS/MS). Other major protein bands appeared on trypsin activity staining included phosphorylase, glyceraldehyde‐3‐phosphate dehydrogenase, and creatine kinase with molecular mass of 95 and 35 to 40 kDa, respectively. But, these 3 proteins did not show true trypsin inhibitory activity. Trypsin inhibitory activity of fractionated sarcoplasmic proteins showed good stability, with >80% activity retained at 60 °C and up to 0.6 M NaCl. TB‐SP showed the highest inhibitory activity against autolysis of washed threadfin bream mince at 65 °C. Addition of 0.5% or 1% TB‐SP improved textural properties of threadfin bream surimi gels preincubated at 37 or 65 °C followed by heating at 90 °C. Therefore, TB‐SP could be a promising protein ingredient for enhancing surimi gel texture.     

Trypsin Inhibitory Activity and Gel‐Enhancing Effect of Sarcoplasmic Proteins from Common Carp

Abstract: Proteinase inhibitory activity of sarcoplasmic protein (SP) extracted from common carp (Cyprinus carpio) muscle and its gel‐improving ability were investigated. SPs displayed 89% and 54% inhibitory activity toward trypsin at 40 and 65 °C, respectively. Protein bands with molecular mass of 69, 50, 44, 41, and 35 kDa appeared on trypsin inhibitory activity staining under nonreducing condition when incubated at 40 °C, while 2 protein bands at 54 and 35 kDa were observed at 65 °C. Addition of SP at 0.18 g protein/100 g increased textural properties of threadfin bream surimi gel. However, when SP was added in combination with various CaCl₂ concentrations (0.1% to 0.5%) it did not further improve textural properties as compared to the addition of SP alone. Retention of myosin heavy chain of threadfin bream surimi was greater with the addition of SP. These results indicated that the gel‐enhancing effect of common carp SP was due to the inhibitory activity toward endogenous trypsin‐like proteinases in threadfin bream surimi. Practical Application: Sarcoplasmic protein from common carp muscle could be used as a functional protein ingredient that minimizes muscle proteolysis and improves textural properties of surimi containing trypsin‐like endogenous proteinases.