The protease inhibitory activity of bovine plasma protein (BPP) and its gel strengthening effect on Pacific whiting surimi were compared with E-64 [L-trans-epoxysuccinylleucylamido (4-guanidio) butane], iodoacetic acid (IAA), and a recombinant soybean cystatin (RSC). In terms of inhibitory activity, as low as 1.2 mM E-64, 37.7 mM IAA, or 17.9 mg RSC were equivalent to 1% BPP. To produce the same gel strength as the 1% BPP-treated surimi, 10 times that level of E-64 and RSC were required, while 100 times that level of IAA did not increase the gel stress as effectively. Thus, plasma contributed to enhanced gelation of Pacific whiting surimi by inhibition of fish protease and also by other gel-enhancing factors in the plasma.

Arrowtooth flounder (AF) fillets are known to contain a heat-activated cysteine protease similar to that found in Pacific whiting, which results in soft texture upon cooking. A crude recombinant soy cystatin (CRSC) produced by Escherichia coli, which has been shown to inhibit the protease(s) in Pacific whiting, was introduced into AF fillets by immersion or injection at one of three levels of inhibitory activity: 10 times less than, equal to, or 10 times greater than that of a 20% bovine plasma protein (BPP) solution, a known inhibitor of AF protease(s). Fillets treated with CRSC or BPP at equal inhibitory strength subsequently exhibited the same degree of protection against textural degradation during cooking. Fillets treated with CRSC at lesser or greater levels of inhibitory activity than those of BPP exhibited lesser or higher protection, accordingly. As revealed by SDS-PAGE, the outer portion of fillets soaked with inhibitory solutions was more effectively protected than the inner portion. Such differences between the outer and inner portions of the fillets were not evident when inhibitory solutions were injected into the fillets.

A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli. An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium. Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin. The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB. Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation. The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation. This study introduced a potential new method for fermentation production of cystatin.

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