*3.1. Sample Collection*

*Cystoseira compressa* samples were collected off the south coast of the island Ciovo in the ˇ Adriatic Sea from May to September 2020 (43.493389◦ N, 16.272505◦ E). Sampling was done throughout a lagoon at 25 points in a depth range of 20 to 80 cm. The sea temperature and salinity were measured during sampling using a YSI Pro2030 probe (Yellow Springs, OH, USA). A sample of this species is deposited in the herbarium at the University Department of Marine Studies in Split.

#### *3.2. Pre-Treatment and Extraction*

Prior to the extraction, harvested algal samples were washed with tap water to remove epiphytes. Samples were then freeze-dried (FreeZone 2.5, Labconco, Kansas City, MO, USA) and ground. Based on the previous research [46] seaweeds were extracted using MAE in the advanced microwave extraction system (ETHOS X, Milestone Srl, Sorisole, Italy). Seaweeds were mixed with 50% ethanol, using 1:10 ( *w*:*v*) algae to solvent ratio and extracted for 15 min at 200 W and 60 ◦C. The extracts were further centrifuged at 5000 rpm for 8 min at room temperature and the supernatant was filtered. The ethanolic solvent was evaporated at 50 ◦C and the rest of the extracts freeze dried.

#### *3.3. Determination of Total Phenolics, Total Tannins and Antioxidant Activity*

The crude algal extracts were dissolved in 50% ethanol prior to analyses in the concentration of 20 mg/mL. Folin–Ciocalteu method [47] was used for determining the TPC. Briefly, 25 μL of the extract was mixed with 1.5 mL distilled water and 125 μL Folin– Ciocalteu reagent. The solution was stirred and 375 μL 20% sodium carbonate solution and 475 μL distilled water was added after one minute. Samples were left in the dark at room temperature for 2 h. The absorbance was read using a spectrophotometer (SPECORD 200 Plus, Edition 2010, Analytik Jena AG, Jena, Germany) at 765 nm. Results were expressed as gallic acid equivalents in mg/g of freeze-dried extract (mg GAE/g).

The TTC was measured according to Zhong et al. [36] with some modifications. Briefly, 25 μL of the sample, 150 μL 4% ( *w*/*v*) ethanolic vanillin solution, and 25 μL 32% sulfuric acid (diluted with ethanol) were added to the 96-well plate and mixed. The plate was incubated for 15 min at room temperature and absorbance was read at 500 nm using the microplate reader (Synergy HTX Multi-Mode Reader, BioTek Instruments, Inc., Winooski, VT, USA). The TTC results were expressed as mg catechin equivalents per g of dried extract (mg CE/g).

The reducing activity was measured as FRAP (ferric reducing/antioxidant power) [48]. Briefly, 300 μL of FRAP reagen<sup>t</sup> solution was pipetted into the microplate wells, and

absorbance at 592 nm was recorded. Then, 10 μL of the sample was added to the FRAP reagen<sup>t</sup> and the change in absorbance after 4 min was measured. The change in absorbance, calculated as the difference between the final value of the absorbance of the reaction mixture after a certain reaction time (4 min) and the absorbance of FRAP reagen<sup>t</sup> before sample addition, was compared with the values obtained for the standard solutions of Trolox. Results were expressed as micromoles of Trolox equivalents per liter of extract (μM TE).

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability of extracts was also measured in 96-well microplates [49]. DPPH radical solution with the initial absorbance of 1.2 (290 μL) was pipetted into microplate wells, and absorbance was measured at 517 nm. Then, 10 μL of the sample was added to the wells and the decrease in the absorbance was measured after 1 h using the plate reader. The antioxidant activity of extracts was expressed as DPPH radical inhibition percentages (% inhibition).

The oxygen radical absorbance capacity (ORAC) method [50,51] was performed to determine the antioxidant capacity of extracts by monitoring the inhibition of the action of free peroxyl radicals formed by the decomposition of 2,2-azobis (2-methylpropionamide)- dihydrochloride (AAPH) against the fluorescent compound fluorescein. Briefly, 150 μL of fluorescein and 25 μL of the sample in 1:200 dilution (or Trolox in the case of standard compound, or puffer in the case of blank) were pipetted into microplate wells and thermostated for 30 min at 37 ◦C. After 30 min, 25 μL of AAPH was added and measurements were performed at excitation and emission wavelengths of 485 and 520 nm every minute for 80 min. The results were expressed as μM of Trolox Equivalents (μM TE).

#### *3.4. Determination of the Antimicrobial Activity*

The foodborne pathogens *Escherichia coli* ATCC 25922, *Salmonella enteritidis* ATCC 13076, *Enterococcus faecalis* ATCC 29212, *Listeria monocytogenes* ATCC 7644, *Staphylococcus aureus* ATCC 25923, and *Bacillus cereus* ATCC 14579 were used in this study.

The microdilution method was used to determine the extracts' MICs against foodborne pathogens. The extracts were dissolved in 4% DMSO (10 mg/mL) and diluted with Mueller– Hinton broth (MHB). Then, 100 μL of the mixture was added to the first well of the 96-well microtiter plate. Two-fold dilutions were done in the next wells (10–0.16 mg/mL). The 50 μL of prepared inoculum (1 × 10<sup>5</sup> colony forming units (CFU)/mL determined by using the growth curves of bacteria in the *log* phase) was added to each well and plates were mixed on a microtiter plate shaker for 1 min at 600 rpm (Plate Shaker-Thermostat PST-60 HL, Biosan, Riga, Latvia). Positive control (50 μL of inoculum and 50 μL of broth media), negative control (50 μL of broth media and 50 μL of extract), blank (100 μL of broth media) and 4% DMSO were also tested. After 24 h of incubation, 20 μL of the indicator of bacterial metabolic activity, 2-*p*-iodophenyl-3-*p*-nitrophenyl-5-phenyl tetrazolium chloride (INT, in 2 mg/mL concentration) was added to each well. Plates were mixed on a plate shaker and incubated for 1 h in the dark. MIC values were read visually as the lowest concentration of the extract at which there was no detection of bacterial growth seen as the reduction of INT to red formazan [52].

MBC of the seaweed extracts was determined as the lowest concentration at which no microbial growth was detected on agar plates after subcultivation of bacterial suspension pipetted from wells where MIC was determined and from wells with higher extract concentrations [53].

#### *3.5. Compound Analysis by UPLC-PDA-ESI-QTOF*

Dried extract (3 mg) of algae was dissolved in 1 mL of MeOH/H2O 1/1 *<sup>v</sup>*/*<sup>v</sup>*. The analysis of compounds from algae was carried out with the use of an ACQUITY Ultra Performance LC system equipped with a photodiode array detector with a binary solvent manager (Waters Corporation, Milford, MA, USA) series with a mass detector Q/TOF micro mass spectrometer (Waters) equipped with an electrospray ionization (ESI) source operating in negative mode at the following conditions: capillary voltage, 2300 kV; source temperature, 100 ◦C; cone gas flow, 40 L/h; desolvation temperature, 500 ◦C; desolvation gas

flow, 11,000 L/h; and scan range, *m*/*z* 50–1500. Separation of individual compounds was carried out using an ACQUITY UPLC BEH Shield RP18 column (1.7 μm, 2.1 mm × 100 mm; Waters Corporation, Milford, MA, USA) at 40 ◦C. The elution gradient test was carried out using water containing 1% acetic acid (A) and acetonitrile (B), and applied as follows: 0 min, 1% B; 2.3 min, 1% B; 4.4 min, 7% B; 8.1 min, 14% B; 12.2 min, 24% B; 16 min, 40% B; 18.3 min, 100% B, 21 min, 100% B; 22.4 min, 1% B; 25 min, 1% B. The sample volume injected was 2 μL and the flow rate used was 0.6 mL/min. The compounds were monitored at 280 nm. Integration and data elaboration were performed using MassLynx 4.1 software (Waters Corporation, Milford, MA, USA) [54].
