We did not try to inoculate B. subtilis after L. bulgaricus was preinoculated.
In our pretest, we found that pH of the fermented red
beans dropped from 6.75 to 5.30 when the steamed red beans were
incubated with L. bulgaricus for two days, and the resultant (acidified
red beans) inhibited the growth of B. subtilis (data not shown).
Our previous study showed that B. subtilis-fermented red beans
contained a fibrinolytic enzyme, a subtilisin-like serine protease,
and its fibrinolytic/caseinolytic activity (using fibrin/casein as a
substrate) was comparable to that of commercial nattokinase
(Chang et al., 2012). Therefore, the fermented red beans were
assessed for their fibrinolytic activity to evaluate the effect of the
inoculation method on the production of the fibrinolytic enzyme.
The red bean sample with the highest fibrinolytic activity was obtained
at 96 h after simultaneous inoculation of the two cultures
(Table 1).
For rest of the study, two starter cultures were inoculated in
steamed red beans simultaneously.