2.7. Antimicrobial activity assay
Four hundred microlitres of LPOS (5.6 U/ml LPO, 1.25 mM KSCN,
0.25% Glc, and 0.87 U/ml GO) were mixed with 50 ll of diluted CE
solutions or b-carotene solution. After adding 50 ll of 107 cfu/ml S.
enteritidis solution, the mixture was incubated at 30 C for 2 h.
Thereafter, 100 ll of the LPOS-treated bacterial suspension, diluted
to countable cell number with 0.15 M NaCl, was streaked onto a
DHL agar plate and incubated at 37 C for 24 h. The number of bacterial
colonies formed on the plate was counted. Control was the
treatment using Milli-Q water instead of the diluted CE solution
or b-carotene solution, and LPOS. Antimicrobial activity of LPOS
was expressed as log N0/N1, where N0 = cfu/ml of control and
N1 = cfu/ml of sample.
2.8. Storage stability of LPOS antimicrobial activity in the presence of
b-carotene
Five kinds of LPOS solutions, with/without b-carotene, were
prepared in the composition shown in Table 1. The LPOS solutions
were incubated at 30 C for 6 h. Then, 450 ll of the solution were
added to 50 ll of 107 cfu/ml S. enteritidis and the bacteria-containing
suspension were incubated at 30 C for 2 h. The counting of
bacteria colonies on a DHL agar plate and the estimation of antimicrobial
activity were conducted as stated above.
2.9. Statistical analysis
Data are presented as means ± standard error of the mean
(SEM). Statistical significance was determined using the GraphPad
Prism statistical software (San Diego, CA, USA). The ANOVA analysis
and the Turkey multiple comparison test were used to determine
the statistical significance. P-values of less than 0.05 were
defined as significant unless otherwise stated.