## 2 Materials and Methods
### Antibacterial Screening
#### 2.3.2 Microdilution Antibacterial Assay  
The MICs of the extracts were determined by a serial dilution method in 96-well sterile microtiter plates. The antimicrobial activity bioassays were optimized according to the requirements and characteristics of each bacterial species. All strains (except the AAB) were grown up to the exponential phase (OD600 = 0.5) in MH broth with agitation at 37 \({}^{\circ}\)C. After that, the cells were centrifuged (2500 rcf for 10 min.) and resuspended in sterile MH broth concentrate (2.5X) at an OD600 of 0.1 (_K. pneumoniae_, _S. dysenteriae_ and _Staphylococcus_ species) or 0.25 (_E. coli_ and _P. aeruginosa_) and mixed with the different extracts at a concentration of between 1.0 and 240 mg gallic acid equivalents per mL (GAE/mL). Briefly, 44 \(\mu\)L of cell suspension (OD600 = 0.1 or 0.25) and 66 \(\mu\)L of the extract were added to each well. The final inoculum contained approximately 2 \(\times\) 10\({}^{5}\) CFU/mL in a total volume of 110 \(\mu\)L/well of MH (1X). The microtiter plates were mixed and incubated under aerobic conditions at 37 \({}^{\circ}\)C for 24 h and then examined using a binocular microscope and a spectrophotometer.  
The AAB strains were grown in YPD broth (up to OD600 = 1.0) with agitation at 28 \({}^{\circ}\)C. The cells were concentrated by centrifugation at 2500 rcf for 10 min. and resuspended in GYC broth concentrate (2.5X) at an OD600 of 1.25 and mixed with the extracts at a concentration of between 1.7 and 400 mg GAE/mL. Briefly, 44 \(\mu\)L of the cell suspension in GYC and 66 \(\mu\)L of the extract were added in each well. The microtiter plates were mixed and incubated under aerobic conditions at 28 \({}^{\circ}\)C for 24 h, and the analysis was performed using a bromo cresol purple (BCP, AppliChem, ITW Reagents, Castellar del Valles, Spain) alkaline solution (0.1% \(w/v\) BCP + potassium hydroxide 0.05 M). For that, 8 \(\mu\)L/well of the BCP solution was added and after one minute the color of the reaction was observed [positive reaction (acid) = yellow, negative reaction (alkaline) = violet].  
Positive controls (containing bacteria without extracts) and negative controls (containing extracts without bacteria) were included in each microplate of each experiment. The MIC was determined as the lowest concentration of extracts that inhibited the visual growth of the bacterial culture on the microplate. The spectrophotometric analysis (except the AAB) was determined using a microdilution automatic reader at a wavelength of 600 nm, and each test sample was considered negative when the bacteria growth displayed 90% inhibition compared to the positive control. In addition, the MBC was determined via the serial subcultivation of 10 \(\mu\)L aliquots of each well in microtiter plates containing 90 \(\mu\)L of fresh MH broth per well and further incubation for 24 to 48 h at 37 \({}^{\circ}\)C. The lowest concentration with no visible growth (at the binocular microscope) and the lowest determined by spectrophotometric analysis was defined as the MBC. In the case of AAB, the MBC was determined with fresh GYC broth at 28 \({}^{\circ}\)C and the evaluation was performed with BCP alkaline solution. Each sample was tested in triplicate in separate experiments. Figure 1 illustrates the whole procedure.