*3.3. Antibacterial Activity*

The antibacterial activity of PEP against five representative foodborne pathogens (*C. jejuni*, *S. typhimurium*, *E. coli*, *S. aureus*, and *L. monocytogenes*) was evaluated. As shown in Figure 1, all extracts were active against at least one of the pathogens studied, and their antibacterial activity was related to the drying procedure used for their conservation. PEP exhibited different levels of growth inhibition against the foodborne pathogens evidencing a strain-dependent effect. Among all the studied powders, the most relevant antibacterial activity was observed for FD extract. In fact, this extract inhibited significantly (*p* < 0.05) the growth of all the bacteria studied except *E. coli* strain. This extract showed a growth inhibition range between 22–52%, depending on the bacterial strain. VD 60 ◦C extract inhibited the growth of three of the five foodborne bacterial strains (*C. jejuni*, *S. aureus*, and *L. monocytogenes*), compressing a growth inhibition range of 17–36%. VD 40 ◦C and VD 80 ◦C inhibited the growth of two bacterial strains (*C. jejuni*-*L. monocytogenes*, and *E. coli*-*L. monocytogenes*) with an inhibition range of 46–58%, and 26–46%, respectively. Otherwise, SPD extract inhibited only the growth of *L. monocytogenes* strain (17% of inhibition). Taking into account the nature of the microorganism, the results showed that *L. monocytogenes* was inhibited by all the PEP in the range of 17%–46%, regardless of the drying process used (Figure 1).

**Figure 1.** Effect of PEP (1 mg/mL) on foodborne bacteria growth after 24 h of incubation. Results represent the percentage of growth inhibition respect to the untreated control (100% of growth) and are expressed as mean ±SD (*n* = 3). Bars marked with asterisk indicate significant growth inhibition respect to the control by *t*-test (*p* ≤ 0.05). Freeze drying (FD); Vacuum drying 40 ◦C (VD 40 ◦C), 60 ◦C (VD 60 ◦C), 80 ◦C (VD 80 ◦C); Spray drying (SPD).

*C. jejuni*, the leading cause of bacterial foodborne diarrheal illness worldwide, was also inhibited for three of the powders used (FD, VD 40 ◦C, and VD 60 ◦C) in a range of 22–58%. FD and VD 60 ◦C (27–36%) affected the growth of *S. aureus*, while *S. typhimurium* and *E. coli* were the bacterial strains with the lowest sensitivity to the all PEP. The different antibacterial activities by PEP against foodborne bacterial strains may be related to the different composition of phenolic compounds of each sample (Table 2), assuming that it is generally accepted that phenolic compounds, present in plant extracts, play a mandatory role in their antibacterial effects [28]. However, as can be deduced from Table 2, it is not the total concentration of phenolic compounds present in the sample, which determines its antibacterial effect, but rather the presence of certain specific polyphenolic compounds in the extract. In this regard, FD was the most active bactericidal extract (Figure 1), showed a significant higher concentration of quercetin-3-*O*-galactoside (hyperoside). Hyperoside is a flavonol glycoside with variety of biological activities, including anti-inflammatory, antioxidant, and antimicrobial activities [29–31]. Its antibacterial effect has been demonstrated both, against gram-negative bacteria such as *P. aeruginosa* [32] and against gram-positive bacteria, such as *S. aureus* [33]. The results obtained in this work suggest that the hyperoside could be involved in the antimicrobial effect observed, since this compound has a significantly higher concentration in the powder obtained by FD, while the rest of the phenolic compounds identified are in concentrations similar or lower than those obtained for the rest of the extracts (Table 2). Apparently, the antibacterial effect of PEP used to be more effective against gram-positive bacteria [14]. This behavior is influenced by differences in the cell membrane constituents. Gram-positive bacteria contain an outer peptidoglycan layer, which is an ineffective permeability barrier; meanwhile gram-negative bacteria have outer phospholipidic membrane carrying structural lipopolysaccharide components, which represent an obstacle for polyphenolic compounds to enter the cell cytoplasm [14,34]. This pattern is also observed in our work, except for *C. jejuni*. Although it is a gram-negative bacterium, *Campylobacter* lacks many of the genetic regulatory networks found in other gram-negative bacteria that allow them to respond to, and cope with, adverse conditions [35]. Accordingly, we have previously demonstrated that *Campylobacter* can be significantly inhibited by different polyphenolic compounds [22,36]. Therefore, antibacterial activity of PEP could be modulated depending of the drying procedure used. The drying process involves several variables, which can change the polyphenolic composition of the extract, resulting in a modified antibacterial response.
