Nutrient broth

The S. mutans ATCC 25175 strains used in this study were selected from culture on blood agar plates incubated at 37 °C for 48 h with a CO₂ pack for anaerobic conditions. The S. mutans overnight culture was made in a BHI (brain–heart infusion) nutrient broth (Biolife, Italiana Srl) (4012302) at 37 °C for 18 h to obtain a 10⁹ CFU/mL bacterial suspension.

Microbiological assays were performed in accordance with the ISO 7932:2005 method. In brief, 10 g of each sample and 90 mL (1:10 (w/w)) of sterilized buffered peptone water (BPW; Oxoid, UK) were placed in a sterile stomacher bag and homogenized for 3 min at 230 rpm using a peristaltic homogenizer (BagMixer^®400 P, Interscience, Saint Nom la Bretèche, France). Subsequently, the homogenized samples were submitted to ten-fold serial dilutions in BPW followed by spread-plating on Mannitol Egg Yolk Polymyxin Agar (MYP; Thermo Scientific, Waltham, MA, USA) and incubated at 30 °C for 48 h. Suspected colonies were counted and randomly picked out (up to five isolates) for the evaluation of hemolytic activity on Trypticase Soy Agar 5% sheep blood (Biolife, Monza, Italy). Confirmatory biochemical assessment of the isolates was carried out by means of a Vitek device (bioMerieux, France), according to the manufacturer’s instructions. Harvested colonies were then subcultured in nutrient broth (Biolife, Monza, Italy), and incubated at 30 °C for 4 h for subsequent real-time PCR.

The antimicrobial activity of different OLE concentrations was evaluated against B. cereus, Salmonella enterica, and Escherichia coli (Di3A microbial collection, University of Catania), representative for milk spoilage, process hygiene indicator, and pathogenic microorganism, respectively.
Bacterial strains were cultured in Nutrient Broth (Biolife Italiana S.r.l., Milano, Italy) for 24–48 h and then inoculated to a final cell concentration of 10⁶/mL into 20 mL of melted Nutrient Agar (Biolife Italiana S.r.l.) cooled at 45 °C, gently mixed, and poured into sterile Petri plates. After solidification, wells were made on agar plates by using a sterile cork borer (5 mm diameter) and filled with 60 µL of pure (at 1.44 mg/mL oleuropein) and diluted extract (1:2 and 1:5 v/v, at 0.72 and 0.29 mg/mL oleuropein, respectively). Control plates were made filling the wells only with sterile distilled water (SDW). Plates were incubated at 30 °C (B. cereus) and 37 °C (S. enterica and E. coli) for 48 h. The inhibitory effect of the extract against the tested bacterial strains was assessed measuring (mm) the inhibition halo (no bacterial growth) around the well. Analyses were carried out in triplicate.

Antibacterial activities of C. ragusina L. CRE extract, fractions and isolated compounds against Gram-negative A. baumannii Durn (Ćurković-Perica et al., 2015 (link)) and Gram-positive S. aureus ATCC 25923 were tested using modified Clinical and Laboratory Standards Institute (CLSI), broth microdilution (BD) using 2,3,5-triphenyltetrazolium chloride (TTC) (Lee et al., 2007 ). The TTC-BD were performed according to the guidelines of the CLSI using 96-well microplates (Clinical Laboratory Standards Institute [CLSI], 2007). The bacteria were grown on nutrient agar (Biolife, Milan, Italy) for 16 h at 36 ± 0.1°C to obtain the cultures in log phase of growth. The bacterial biomass was then suspended in sterile NaCl (0.85% v/v) to give turbidity equivalent to the McFarland 0.5 standard. Bacterial suspension (0.1 mL) was transferred to a tube containing 9.1 mL nutrient broth (Biolife) and 0.8 mL 0.05% TTC to give an inoculum density of 1 × 10⁶ Colony Forming Units (CFU)/mL. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined in triplicates. The final concentrations for MIC and MBC determination of samples were 1.9–4000 μg/mL. Other data on antibacterial experiments are available in the Supplementary Material.

For acid tolerance determination, several pH grades were prepared by hydrochloric acid solution (Merck, Germany) 5N to pH 2.0 and 4.0 in Phosphate-Buffered Saline (PBS). The isolates were incubated in nutrient broth (Biolife, Italy) for 18 h at 37 °C, then cell pellet was harvested and washed in PBS, resuspended in both pH solutions, including 2 and 4, and incubation was done for 4 h, 37 °C. Plate counts on nutrient agar at 0 h and 4 h were used for assessment of survivability according to this equation: $$\text{Survival}\text{rate}\left(\%\right)=\left(N1/N0\right)\times 100,$$ where N1 and N0 represent (log cfu/ml) count of selected species after and before treatment respectively^{37 (link)}.

Bacterial strains used were two strains of Gram-negative bacteria, E. coli O157:H7 ATCC 51659 and P. aeruginosa ATCC 27853 and two strains of Gram-positive bacteria: S. aureus ATCC9144 and B. subtilis ATCC 6051. One strain of unicellular fungi (yeast) was used: Candida albicans (C. albicans) ATCC 90028 and also two multicellular filamentous fungi: F. oxysporum and A. fumigatus.Antibiotics were purchased commercially. All media were prepared and sterilised by autoclaving at 121 °C for 15 min. Nutrient agar (Merck, Germany), nutrient broth, Trypticase soy broth and agar (Biolife, Italy) were used for bacteria. Sabouraud dextrose agar (SDA) and SD broth were used for C. albicans. Potato dextrose agar (PDA) medium was used for the filamentous fungal species (F. oxysporum and A. fumigatus). The pH value of the PDA and SDA is 5.6 ± 0.2 at 25 °C. The pH values of the Nutrient agar, broth, and trypticase soy broth media are (6.9, and 7.3 ± 0.2) respectively. All chemical compounds to be tested were dissolved in DMSO. The PH of the tested compounds was neutral.