A total of 30 experimental runs were generated based on CCD
five-level-four-factors design for optimization of conditions as
shown in Table 1. Analysis of variance (ANOVA) was estimated
with Design Expert software (Version 7.0, Stat-Ease Inc.,
Minneapolis, MN, USA).The pH of each fermented milk sample
(50 mL) was adjusted to 4.3 with 1M HCL. After centrifugation
at 3000 × g for 20 min, supernatant was collected and the
pH was re-adjusted to 7.0 with 1M NaOH and re-centrifuged
at 3000 × g for 20 min. The supernatant was passed through a
0.45 m membrane filter and stored at −20 °C until assay. The
results were fitted into a second-order polynomial equation by
a multiple regression technique.