Fig. 1. Curves of surface response of chitosan amounts obtained from C. elegans cultivated
in CSL-based media at different pH values and concentrations of corn steep liquor (CSL).
C.E.V. de Oliveira et al. / International Journal of Food Microbiology 171 (2014) 54–61 57
formation of carbonized residues. The third stage began at 238 °C and
correlated with the consumption of the formed carbonized residue.
The DSC curve registered two thermal events: the first event corresponded
to values between 28 °C and 143 °C and represented an endothermic
event related to evaporation of water, and the second event
corresponded to an endothermic peak associated with the presence of a
weak intramolecular hydrogen bond in the tested CHI and degradation
of this polymer.
3.3. In vitro antifungal effects of CHI
CHI from C. elegans UCP 542 exhibited an MIC value (for the inhibition
of spore germination) of 15 mg/mL against both B. cinerea
URM 5627 and P. expansum URM 331. Different concentrations of
the obtained CHI (1/2 MIC, 3.75 mg/mL; MIC, 7.5 mg/mL; 2 x MIC,
15 mg/mL) were chosen for assaying the inhibition rates of mycelial
growth and spore germination and its application on grapes. Over
7 days of incubation, CHI at all tested concentrations strongly
inhibited the mycelial growth of the test post-harvest pathogenic
fungal strains when compared to the control assays, with inhibition
rates ranging from 65.2 to 94.6% for B. cinerea and from 68.6 to
96.7% for P. expansum (Table 2).
The application of CHI from C. elegans UCP 542 at the different tested
concentrations also revealed high rates of inhibition of spore germination
for both assayed fungi (Table 2). The rates of spore germination
inhibition of the test strains at all assayed CHI concentrations were
greater than 78% compared to the number of spores germinated in the
control test (approximate number of germinated spores, B. cinerea:
4.6 × 105 spores/mL, P. expansum: 6.4 × 105 spores/mL). CHI at 2 ×
MIC presented the highest rates of spore germination inhibition for
both B. cinerea and P. expansum. Applying the CHI at MIC (7.5 mg/mL)
caused sharp morphological changes in the spores of B. cinerea and
P. expansum and disrupted their structures. SEM analyses of B. cinerea
and P. expansum spores exposed to CHI (Fig. 3B–D) revealed changes in
the form and structure, with increased wilting and deepening of typical
ridges. When not exposed to CHI, the B. cinerea and P. expansum spores
exhibited a normal globular, elliptical structure with smooth, typical
ridges (Fig. 3A–C).