## 3 Results and Discussion
### Influence of C. cardunculus Extracts in Melanin Bioproduction  
The _P. putida_ ESACB 191 strain is responsible for the brownish color observed in the rind of cheeses, which has been identified as melanin [16]. Therefore, the study of the influence of RA, CA, CA: RA, CL, CI, and CL: CI, on melanin bioproduction was tested for _P. putida_ ESACB 191 strain (Figure 3). \begin{table}
\begin{tabular}{l c c c} \hline \hline \multirow{2}{*}{**Compounds Tested**} & \multicolumn{3}{c}{**Log CFU/mL Reduction**} \\ \cline{2-4}  & **24 h** & **48 h** & **72 h** \\ \hline Chlorogenic acid (CA) & 3.22 \(\pm\) 0.04 a  & 2.32 \(\pm\) 0.05 a  & 3.80 \(\pm\) 0.05 a  \\ Rosmarnic acid (RA) & 2.25 \(\pm\) 0.14 b  & 1.32 \(\pm\) 0.13 b  & 1.95 \(\pm\) 0.13 b  \\ Chlorogenic acid-Rosmarnic acid (CA:RA) 50:50 & 2.18 \(\pm\) 0.07 b  & 1.31 \(\pm\) 0.0 b  & 1.83 \(\pm\) 0.07 b  \\ C. _cardunculus_ leaves extract (CL) & 6.20 \(\pm\) 0.45 c  & 5.25 \(\pm\) 0.04 c  & 5.10 \(\pm\) 0.14 c  \\ C. _cardunculus_ inflorescrectract (CI) & 3.31 \(\pm\) 0.13 d  & 3.00 \(\pm\) 0.14 d  & 2.98 \(\pm\) 0.15 d  \\ C. _cardunculus_ leaves extract: inflorescrectrectract (CL) & 2.22 \(\pm\) 0.15 b  & 1.01 \(\pm\) 0.07 b  & 2.49 \(\pm\) 0.00 b  \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Influence of _C. cardunculus_ decoction extracts and standard compounds in _P. putida_ ESACB 191 growth reduction (Log CFU/mL), over the incubation time (24, 48 and 72 h). Different superscript letters in the same line, correspond to statistically different values (\(p\leq 0.05\)). Figure 3A corresponds to the control test that had the optimal conditions for _P. putida_ ESACB 191 to produce melanin as described in the previous study [16]. It was verified, at a 95% confidence level, that there are significant differences in the bioproduction of melanin, except for the CL:CI mixture (Figure 3G) which showed similar melanin bioproduction with the Control (Figure 3A) at 72 h. RA (Figure 3B), CA:RA (Figure 3D), CL (Figure 3E) and CI (Figure 3F) extracts, although they showed differences in the bioproduction of melanin by _P. putida_ ESACB 191 compared to the control (Figure 3A), did not differ from each other for a 95% confidence level. However, the influence of CA (Figure 3C) on melanin bioproduction resulted in significant differences at 24, 48 and 72 h when compared to the control (Figure 3A). Namely, at 48 h there was a drop in the bioproduction of melanin by _P. putida_ ESACB 191 of 50%, for a confidence level of 95%. This tyrosinase inhibitory action by chlorogenic acid agrees with the literature where it is described that chlorogenic acid affects the production of melanin through the inhibition of tyrosinase when converted into metabolites in cells [31; 32]. The growth curves present in the results of Figure 3 help us understand the bioproduction of melanin as a function of the cell viability of _P. putida_ ESACB 191. Thus, it is possible to verify that in the presence of the CL extract there is a break in bacterial growth, but it does not affect the bioproduction of melanin. This means that _P. putida_ ESACB 191 secreted tyrosinase into the culture medium during the initial growth of the bacteria [33; 34; 35; 36]. Figure 3: Influence of _C. cardunculus_ extracts and phenolic acids in melanin bioproduction by _P. putida_ ESACB 191. (**A**). Control; (**B**). Rosmarinic acid; (**C**). Chlorogenic acid; (**D**). 50% Chlorogenic acid + 50% Rosmarinic acid; (**E**). Leaf decoction extract; (**F**). Inflorescence decoction extract; (**G**). 50% Leaves decoction + 50% Inflorescence decoction. The concentration 1 mg/mL was used for all samples tested in Müller–Hinton Broth with 1% of L-tyrosine. Lines represent _P. putida_ ESACB 191 growth curve (Log CFU/mL) and bars represent melanin bioproduction (mg/mL). However, tyrosinase activities are induced in the stationary phase, where they are involved in extracellular melanin production [36]. This explains the reason for melanin production by _P.