Quality improvement of threadfin bream (Nemipterus virgatus) surimi-gel using soy protein as a natural food additive.

Previously, we identified sarcoplasmic serine proteinase (SSP) as a modori-inducing proteinase from threadfin bream belly muscle. In this study, we investigated the autolytic activity of commercial threadfin bream surimi under modori-inducing conditions. High autolytic activity was detected in commercial surimi and was inhibited by a soybean trypsin inhibitor, indicating that SSP still remained in the commercial surimi. The effects of soy protein, defatted soy protein (DSP) and isolated soy protein (ISP), on SSP activity and surimi-gel properties were evaluated. The results showed that the modori phenomenon was induced at 70 °C, and that both DSP and ISP suppressed SSP activity and strengthened the breaking strength and breaking distance of the modori-induced gel. Surimi-gel with DSP performed better on gel whiteness than that of ISP, and 1 g/kg DSP had optimal gel properties. In conclusion, soy protein proved to be a good natural food additive for surimi-gel production of threadfin bream.

Transcriptional regulation of the Japanese flounder Cu,Zn-SOD (Jfsod1) gene in RAW264.7 cells during oxidative stress caused by causative bacteria of edwardsiellosis.

Edwardsiellosis is one of the most important bacterial diseases in fish, sometimes causing extensive economic losses in the aquaculture industry. Our previous studies demonstrated that the Cu,Zn-SOD (sod1) activity has significantly increased in Japanese flounder, Paralichthys olivaceus, hepatopancreas infected by causative bacteria of edwardsiellosis Edwardsiella tarda NUF251. In this study, NUF251 stimulated intracellular superoxide radical production in mouse macrophage RAW264.7 cells, which was reduced by N-acetylcysteine. This result suggests that NUF251 infection causes oxidative stress. To evaluate the regulatory mechanism of Jfsod1 at transcriptional levels under oxidative stress induced by NUF251 infection, we cloned and determined the nucleotide sequence (1124 bp) of the 5'-flanking region of the Jfsod1 gene. The sequence analysis demonstrated that the binding sites for the transcription factors C/EBPα and NF-IL6 involved in the transcriptional regulation of the mammalian sod1 gene existed. We constructed a luciferase reporter system with the 5'-flanking region (-1124/-1) of the Jfsod1 gene, and a highly increased transcriptional activity of the region was observed in NUF251-infected RAW264.7 cells. Further studies using several mutants indicated that deletion of the recognition region of NF-IL6 (-272/-132) resulted in a significant decrease in the transcriptional activity of the Jfsod1 gene in NUF251-infected RAW264.7 cells. In particular, the binding site (-202/-194) for NF-IL6 might play a major role in upregulating the transcriptional activity of the 5'-flanking region of the Jfsod1 gene in response to oxidative stress induced by NUF251 infection. These results could be provided a new insight to understand the pathogenic mechanism of causative bacteria of edwardsiellosis.

Identification of a modori-inducing proteinase in the threadfin bream: Molecular cloning, tissue distribution and proteinase leakage from viscera during ice storage.

Previously we purified and characterized a sarcoplasmic serine proteinase (SSP) from the belly muscle of the threadfin bream as a modori-inducing proteinase. In our attempt to clarify the structure and physiological functions of SSP, we successfully cloned the full-length cDNA of SSP (ORF 726 bp). The deduced amino acid sequence of SSP (241 residues) was highly homologous to fish trypsinogen. The distribution of SSP mRNA and the proteinase activity in the tissue indicated that SSP was mainly synthesized and existed in the digestive system under physiological conditions. After ice storage of the threadfin bream without gutting, a high SSP activity was detected only in the belly muscle because of SSP leaked from the viscera. Therefore, it is desirable to use edible proteinase inhibitor to inactivate the leaked SSP during production of surimi-based products or to take effective measures to prevent the proteinase leakage during post-harvest storage.

Purification and characterization of a sarcoplasmic serine proteinase from threadfin bream Nemipterus virgatus muscle.

A sarcoplasmic serine proteinase (SSP) was purified from threadfin bream (Nemipterus virgatus) belly muscle by ammonium sulfate precipitation and a series of chromatographies including Q-Sepharose, Phenyl Sepharose and Superdex 200. The SSP was purified 1967 folds with a yield of 4.8%. The molecular weight of the SSP was estimated to be 43.5 kDa and 22.5 kDa on SDS-PAGE under non-reducing and reducing conditions, respectively. The N-terminal amino acid sequence of the two protein bands were determined as IVGGYEXQPYSQAHQVSLNSGY and corresponded. It is suggested that the SSP exists as a homodimer. Optimum pH and temperature were 9.5 and 50 °C, using Boc-Val-Pro-Arg-MCA as a substrate. Substrate specificity and effects of inhibitors indicated that the SSP was a trypsin-like serine proteinase. The SSP was responsible for hydrolyzing myosin heavy chain (MHC) and inducing modori phenomenon in the threadfin bream surimi gel. Thus, the SSP was considered as a modori-inducing proteinase.