Changes in proteinase activities of

Changes in proteinase activities of all fish sauce samples
from different treatments during fermentation are
shown in Fig. 1. The activity decreased continuously
as the fermentation time increased (p < 0.05) (Fig. 1).
However, the rate of changes varied, depending on the
salt concentration and spleen level added. No changes in activity were found in the samples containing 20%
spleen and 25% salt in the first 20 days of fermentation
(p > 0.05). The decrease in activity might be due to the
denaturation of enzymes, particularly in the presence
of a high salt content. The loss in activity was also
thought to be due to the inhibition by end-products,
such as amino acid and short chain peptides (Orejana
& Liston, 1982).
Evidently, different spleen levels and salt concentrations
resulted in different proteinase activities
(p < 0.05). At the same level of salt concentration, the
fish sauce with 20% spleen showed the hightest activity,
followed by those with 10% and without spleen, respectively.
Spleen was reported to consist of a large amount
of proteinase (Klomklao et al., 2004). Salt concentrations
directly affected proteolytic activity in the fish
sauce sample. Generally, the activity decreased with
increasing NaCl concentration. The salting out effect
was postulated to cause the enzyme denaturation. A
water molecule is drawn from the proteinase molecule,
leading to the aggregation of those enzymes (Klomklao
et al., 2004). Increased ionic strength caused by high salt
concentration, along with the extended incubation time
at high temperature (37 C), possibly resulted in the increased
denaturation and loss of enzyme activity
(Tungkawachara, Park, & Choi, 2003). The activity of
trypsin-like enzyme in protein hydrolysate, made from
75% fish viscera and 25% salt at 27 C, was only 10%
after 50 days (Gildberg, 1992). From the results, it was
noted that no proteolytic activity of sample without
spleen addition could be observed after 10 days of fermentation
in the presence of high salt (25% salt). Nevertheless,
some proteinase activity of the samples without
spleen was still found in the presence of less salt at every
fermentation time. This indicated the role of endogenous
proteinases in sardine, which play an essential role
in autolysis of this species. Thermostable proteinases
were still active and able to degrade myofibrillar protein
in commercial salted anchovy containing 16–17% salt
(Ishida, Niizelei, & Nagayama, 1994). The activity of
acid proteinases from sardine was reduced by the addition
of 3.42 M salt (Noda & Murakami, 1981). Nevertheless,
Fang and Chiou (1989) reported that salt, up
to 3.42 M, had no effect on pepsin, trypsin or chymotrypsin
activities from tilapia. Thus, salt stability of proteinases
depends upon fish species as well as salt
concentrations. High salt concentration (25%) prolonged
fish sauce shelf life but it inhibited peptidase
activity and hence retarded protein hydrolysis
(Gildberg, 1989). However, salt reduction, from 25%
to 5–15%, accelerated autolysis during fish sauce fermentation
(Sikorski et al., 1995). From the results,
spleen supplementation, as well as salt reduction from
25% to 15–20%, resulted in an increased proteolytic
activity and a retarded loss of proteolytic activity of
the fish sauce sample.