*2.2. Antimicrobial Activity*

The results of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the *C. compressa* extracts against common foodborne pathogens are shown in Table 2. Gram-positive bacteria were more susceptible to seaweed extracts than Gram-negative bacteria. The lowest MIC results were found against *L. monocytogenes*, for June, July, and August samples with the lowest MBC in June, and against *S. aureus* in July and August with the same MBC. There was no difference in MIC and MBC values for *E. coli* among the investigated months. June, July, and August extracts had the lowest MIC values for *S. enteritidis*. The results showed higher antimicrobial activity from June to August when the sea temperature was the highest, against all bacteria.

**Table 2.** Results of the minimal inhibitory concentration (MIC, mg/mL) and minimal bactericidal concentration (MBC, mg/mL) of the seaweed extracts against foodborne pathogens (*n* = 3).


n.d.—not determined

Alghazeer et al. [33] performed microwave-assisted extraction (MAE) on *P. pavonica* and *C. compressa*. Flavonoid-rich extracts (110.92 ± 11.38 mg rutin equivalents /g for *C. compressa*) were tested for antibacterial activity against multidrug-resistant (MDR) isolates of *S. aureus* subsp. *aureus*, *Bacillus pumilus*, *B. cereus*, *Salmonella enterica* subsp. *enteric*, and enterohemorrhagic *E. coli* using the well diffusion method, MIC and MBC. *Cystoseira compressa* extract showed stronger antibacterial activity than *P. pavonica* with inhibition zones against 14 tested isolates. The largest inhibition zones were 20.5 mm for *S. aureus* and *B. cereus*, 31 mm for *S. enterica* and 17 mm for *E. coli*. Furthermore, *C. compressa* extract had the lowest MIC (31.25 μg/mL) and MBC (62.5 μg/mL) values against *S. aureus* and *S. enterica*. Against *B. cereus*, it had an MIC value of 62.5 μg/mL and an MBC value of 125 μg/mL. The highest MIC (125 μg/mL) and MBC (500 μg/mL) values were found against *E. coli*. Maggio et al. [28] evaluated the antibacterial activity of eight brown seaweeds, six belonging to the genus *Cystoseira* (including *C. compressa*) and two belonging to the Dictyotaceae family, against *E. coli*, *Kocuria rhizophila*, *S. aureus* and a toxigenic and MDR *S. aureus* using the disk diffusion method. None of the seaweed extracts inhibited the growth of *E. coli*. *Cystoseira compressa* and *Carpodesmia amentacea* extracts showed antibacterial activity against *K. rhizophila*, *S. aureus* and MDR *S. aureus*. Abdeldjebbar et al. [34] tested the antibacterial effect of *C. compressa* and *P. pavonica* acetonic extracts against *E. coli* and *S. aureus*. The antibacterial activity was measured by disk diffusion method and MIC determination. *Cystoseira compressa* extract had 14 mm inhibition diameter for *E. coli*, showing better antibacterial activity than *P. pavonica* (12 mm). However, MIC values were not detected for *C. compressa* against both bacteria. *Padina pavonica* extract had an MIC of 50 μL for tested strains. Both extracts had a 10 mm inhibition diameter for *S. aureus*. The authors also tested

the synergy of these two extracts at a 1:1 ratio. The mixture showed significant synergistic effect against *E. coli* and *S. aureus* with 16 and 12 mm inhibition diameters, respectively. The antibacterial activity of a *C. crinita* cold methanolic extract was evaluated by the disk diffusion method [18]. The extract showed the highest inhibition zones for *E. coli*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Bacillus subtilis*, *S. aureus* and *Streptococcus aureus*, with 10.5, 12.8, 10.2, 12.6, 13.3 and 11.2 mm, respectively.

*Cystoseira compressa* extracts [31] showed moderate activity against *E. coli*, *S. aureus*, *Streptococcus epidermidis*, *E. faecalis*, *Enterobacter cloacae*, *Klebsiella pneumonie*, *B. cereus* and *P. aeruginosa*. In this study, the authors found the lowest MIC value of 32 μg/mL for both methanolic extract against *S. epidermidis* and chloroform extract against *E. cloacae*. Dulger and Dulger [21] tested *C. compressa* water and ethanol extracts against methicillin-resistant *S. aureus* (MRSA). Ethanol extract had the lowest MIC of 3.2 mg/mL and MBC of 6.3 mg/mL.

In the above-mentioned studies, the chemical content of the investigated algae was not correlated with the antimicrobial activity, however, it is evident that *Cystoseira* spp. shows some potential to be used nutraceuticals and therapeutic purposes.

#### *2.3. Chemical Analysis by UPLC-PDA-ESI-QTOF*

A quali-quantitative analysis of the polar compounds from *C. compressa* extracts was achieved by LC-ESI-QTOF-MS analysis in negative ion mode. The base peak chromatograms obtained are shown in Figure 3. A total of 49 compounds were identified and the results are shown in Table 3, along with their retention time, observed and theoretical *<sup>m</sup>*/*<sup>z</sup>*, error (ppm), score (%), molecular formulae and in source fragments. In all cases, the score remained higher than 90% and the error lower than 5 ppm. All the compounds we tentatively identified according to Bouafif et al. [35] who previously found most of them in *Cystoseira* and PubChem database. Furthermore, the amount of each compound is expressed as a percentage calculated based in the areas for each extract.

**Figure 3.** Chromatograms of the HPLC-qTOF-MS analyses of *C. compressa*.




**Table 3.** *Cont.*

The most dominant compound tentatively identified was oleic acid (C18:1n-9) with a content more than 15% in all tested samples, highest in May. The other two dominant compounds were palmitoleic acid (C16:1n-7) and palmitic acid (C16:0) also showing highest content in the May extract. Except for highly represented fatty acids, ω-3 eicosapentaenoic acid (EPA) was also found, with the highest content in July.

Low molecular weight phenolic compounds were not identified. This does not confirm that the phenolics are not present, but the main phenolics in algae are probably present as tannins (phlorotannins) that cannot be determined by HPLC-ESI-TOF-MS because they cannot be ionized due to their high molecular weight. Maggio et al. [28] reported citric acid, isocitric acid, vanillic acid methyl ester, vanillic acid sulfate, gallic acid, dihydroxybenzoic acid, 2-hydroxy-6-oxo-6-(2-hydroxyphenoxy)-hexa-2,4-dienoate, phloracetophenone, bromo-phloroglucinol, vanillylmandelic acid and exifone in *C. compressa*. The compounds were identified without quantification. Previously, vanilic acid, hydroxybenzoic acid, gallocatechin, carnosic acid, phloroglucinol, and hydroxytyrosol 4-*O*-glucoside were identified as main phenolics in fucoidan algae *Sargassum* sp. [36].

Jerkovi´c et al. [37] investigated fucoidal brown alga *Fucus virsoides* and found 42.28% oleic acid, 15.00% arachidonic acid and 10.51% myristic acid in its fatty acid composition. The authors used high performance liquid chromatography–high-resolution mass spectrometry (HPLC-ESI-HRMS) to determine the composition of less polar non-volatile compounds. The major compounds tentatively identified belonged to five groups, steroids, terpenoids, fatty acid glycerides, carotenoids, and chlorophyll derivatives. Fatty acid glycerides were dominant, which is comparable to our study.

Ristivojevi´c et al. [38] identified the bioactive compounds responsible for the radical scavenging and antimicrobial activities of *Undaria pinnatifida* and *Saccharina japonica* methanolic extracts using the high-performance thin layer chromatography (HPTLC)- bioautography assay and ultra-high-performance liquid chromatography (UHPLC)-LTQ-MS/MS combined. They reported eicosapentaenoic, stearidonic and arachidonic acids as major compounds accountable for these activities. Their findings are in accordance with previous reports on PUFAs having antimicrobial activity against bacteria, viruses and fungi [39,40].

PUFAs, such as EPA, docosahexaenoic acid (DHA) and linolenic acid (LNA), showed in vitro antibacterial activity against *Helicobacter pylori*, *S. aureus*, Methicillin-resistant *S. aureus* (MRSA), *Vibrio vulnificus*, and *Streptococcus mutans*, inhibiting bacterial growth or altering their cell morphology [40]. To deactivate microbial cells, PUFAs directly affected the cell membranes, enhanced free radical generation, and increased the formation of cytotoxic lipid peroxides and their bioactive metabolites increasing the leukocytes' and

macrophages' phagocytic action [39]. EPA and DHA extracts showed antimicrobial activity against foodborne pathogenic bacteria, *L. monocytogenes*, *B. subtilis*, Enterobacter aerogenes, *E. coli*, *S. aureus*, *S. enteritidis*, *S. typhimurium*, and *P. aeruginosa* [41]. The authors reported the lowest MIC value of 250 μg/mL for DHA extract against *P. aeruginosa*. A low MIC value of 350 μg/mL was found for EPA extract against *L. monocytogenes*, *B. subtilis* and *P. aeruginosa*, and for DHA extract against *L. monocytogenes* and *B. subtilis*. Besides, Cvitkovi´c et al. [42] investigated the extraction of lipid fractions from *C. compressa*, *C. barbata*, *F. virsoides*, and *Codium bursa*. In agreemen<sup>t</sup> with our results, the dominant fatty acids in all seaweeds were palmitic, oleic and linolenic fatty acids. *Cystoseira compressa* and *C. barbata* had the highest amounts of omega-3 EPA and DHA. *Cystoseira compressa* had 20.35% oleic acid, 17.66% arachidonic acid, 14.86% linoleic acid, 11.92% palmitic acid and 8.72% linolenic acid. Bacteria *S. aureus* can be inhibited by most free fatty acids: Lacey and Lord [43] seeded this bacterium on human skin and then applied LNA to the skin which resulted in the rapid death of the seeded bacteria. EPA (C20:5n-3) was found to successfully inhibit the growth of *S. aureus* and *B. cereus* with a 64 mg/L MIC value [44]. Oleic acid was confirmed in vitro and in vivo to effectively eliminate MRSA by disrupting its cell wall [45].

#### **3. Materials and Methods**
