## 3 Results and Discussion
### Influence of C. cardunculus Extracts on Bacterial Growth
#### 3.2.2 Antimicrobial Activity of _C. cardunculus_ Extracts Using Liquid Method  
_P. putida_ ESACB 191 showed the same response as _Acaligenes faecalis_ ESACB 7 and _P. fluorescens_ ESACB 67 against the antimicrobial action of CA and RA. However, this strain was described by [PERSON] et al. in 2021 [16] as producing melanin and was therefore selected for further testing. To understand whether the inhibition of _P. putida_ ESACB 191 by phenolic acids, detected in the disk diffusion method described above, is influenced by physicochemical properties such as the penetration of antimicrobial agents into the disk, colony forming units (CFU) were used to measure cell viability in the presence of: chlorogenic acid (CA), rosmarinic acid (RA), CA:RA mixture (50:50), _C. cardunculus_ leaves extract (CL), _C. cardunculus_ inflorescence extract (CI) and CL:CI mixture (50:50). To compare the antimicrobial activity, the same concentration of antimicrobial agents (1 mg/mL) was used in both methods. The results of the influence of the samples on the growth of _P. putida_ ESACB 191 in a liquid assay are shown in Figure 2.  
Figure 2: _P. putida_ ESACB 191 growth curves by action of CA—Chlorogenic acid; RA—Rosmarinic acid; CA:RA—50% Chlorogenic acid + 50% Rosmarinic acid, and _C. cardunculus_ extracts: CL—Leaves decoction; CL—Influorescence decoction; CL:CI—50% leaves decoction + 50% inflorescence decoction. The concentration 1 mg/mL was used for all samples tested in Müller–Hinton Broth with 1% L-tyrosine. Figure 2 shows that all samples reduce the growth of _P. putida_ ESACB 191 compared to the control at a 95% confidence level, within the first 24 h of incubation time. CL showed the greatest reduction in bacterial growth at 24 h, followed by CI and CA. RA and the mixtures CA:RA and CL:CI did not show significant differences between them, however they were the ones that reduced the growth of _P. putida_ ESACB 191 less compared with the control, for a confidence level of 95%. Table 3 shows the reduction of the tested compounds in _P. putida_ ESACB 191 growths at 24, 48 and 72 h of incubation. None of the tested compounds showed differences between incubation time periods, but they showed differences between them. CL showed the most significant decrease in bacterial growth, for a 95% confidence level, which corresponds to a mean decrease of 60% in the growth of _P. putida_ ESACB 191. CI decreased bacterial growth by about 33% on average. CA also showed a mean decrease of 33%, however it was statistically different from CI at a 95% confidence level. RA, CA:RA and CL:CI did not show significant differences between them in decreasing the growth of _P. putida_ ESACB 191 over time, however they were the ones that had less influence on the decrease of bacterial growth. Contrary to the results obtained in the antimicrobial activity test in solid medium (disk diffusion method), in the liquid test, the CL and CI extracts were the ones that showed the greatest influence in reducing the growth of _P. putida_ ESACB 191. These results suggest that the disk usage decreases the diffusion capacity of the extracts in the culture medium. The discrepancies between the methods can be explained by the fact that the aqueous extracts tested are mixtures of compounds, some of them with relatively high molecular weight compounds, as in the case of CL 533 \(m/z\). Due to their molecular weight and probably to their poor hydrophilicity, they do not dissolve easily into the agar medium. The compounds present in the extracts have limited migration into the agar, erroneously leading to their non activity as bacteria growth inhibitors [27; 28; 29]. CL has chlorogenic acid and its derivatives as the major component of its chemical constitution (Table 1) and presented an antimicrobial action that resulted in a 66% decrease in the growth of _P. putida_ ESACB 191. This decrease results not only from the presence of chlorogenic acid that showed less antimicrobial activity than CL, but from the synergistic effect of all compounds present in this extract.