4.4.1. Antibacterial Activity

Four bacterial strains: *Staphylococcus aureus* ATCC 29213, methicillin-resistant *Staphylococcus aureus* (SMR), *Escherichia coli* ATCC 25922 and *Salmonella* Enteritidis ATCC 13076, were used as indicator strains for the determination of the antibacterial activity. Exopolysaccharides samples were tested for antibacterial activity by using the well diffusion method and the minimum inhibitory concentration (MIC) using the microdilution method on sterile 96-well plate [64]. Briefly, on Petri dishes containing solid Luria broth (LB) medium previously inoculated with the bacterial inoculum, 6 mm wells were dug in the medium and then sealed with a thin layer of liquefied soft agar to limit the diffusion of the sample under the solid medium. Samples with 50 μL of exopolysaccharides were placed in each well. Sterile distilled water was used as a negative control. The diameters of the inhibition zones were measured after 18 h of incubation at 30 ◦C.

To determine the MIC, bacterial strains were grown in LB medium at 30 ◦C. 10<sup>5</sup> CFU/mL of each bacterial suspension were incubated for 24 h at 30 ◦C in the presence of the test sample at concentrations ranging from 7.81 to 1000 μg/mL. Wells containing the bacterial strain alone were used as a positive control, while wells containing only the culture medium were considered as a negative control. MIC values were determined for the lowest concentration of the extract, showing complete inhibition of bacterial cell growth.

#### 4.4.2. Anti-Candida Albicans Activity

Detection of antifungal activity against *Candida albicans* 10231 was performed using the microdilution method on a 96-well plate, as previously described by Grieco et al. [65]. Briefly, a culture of *C. albicans* (30 ◦C in WB medium) is diluted in the same growth medium to reach 10<sup>5</sup> UFC/mL and the suspension is introduced in each well, with different concentrations of samples between 7.81 and 1000 μg/mL and the plate is incubated 24 h at 37 ◦C. Two controls were included. The negative control corresponds to the growth medium, and the positive control contains *C. albicans* in the growth medium. MIC values were determined as the lowest concentration of the extract showing complete inhibition of *Candida* cell growth.

#### 4.4.3. Inhibition of Biofilm Formation

Biofilm quantification was performed using the crystal violet staining assay on a 96-well polystyrene plate, as demonstrated by Jin et al. [66]. Indeed, a suspension of *Candida albicans* with a final concentration of 10<sup>5</sup> CFU/mL was prepared in the RPMI 1640 culture medium (R8758, Sigma-Aldrich, St. Quentin Fallavier Cedex, France). Fifty microliters of *C. albicans* inoculum was distributed into each well of the microplate in the presence of different exopolysaccharides samples, with concentrations ranging from 7.81 to 1000 μg/mL to have a final volume of 100 μL. The plate was then incubated at 37 ◦C for 24 h. After incubation, crystal violet staining was performed to demonstrate the inhibitory effect of biofilm formation. Briefly, the culture medium was removed and each well was washed twice with PBS buffer (pH = 7.4; 0.1 M). For biofilm fixation, 100 μL of 99% methanol was added. The biofilm was then stained with 0.05% crystal violet for 15 min at room temperature. The excess was removed by two washes with ultra pure water. Purple coloration appeared after the addition of 100 μL of 33% acetic acid and was detected at 630 nm. The percentage of biofilm inhibition was determined by the equation below:

$$\% \text{ inhibition} = [100 - ((A\_{\text{Sample}} - A\_{\text{blank}})/(A\_{\text{Control}} - A\_{\text{blank}}))] \times 100 \tag{1}$$

with ABlank and AControl corresponding to the absorbance of RPMI medium and untreated biofilm at 630 nm, respectively.

## 4.4.4. Cytotoxicity Evaluation

Cytotoxicity EPS samples were tested on mammalian cell line Vero using MTT method according to Medjeldi et al. [67], measuring absorbance at 540 nm. The percentage of cytotoxic effect was calculated using the following equation:

$$\left[ (\text{A}\_{\text{Control}} - \text{A}\_{\text{Sample}}) / (\text{A}\_{\text{Control}}) \right] \times 100. \tag{2}$$

The 50% cytotoxic concentration (CC50), defined as the sample concentration able to reduce the cell viability by 50% when compared to an untreated control, was determined by linear regression analysis from a dose-response curve.

#### 4.4.5. Antiproliferative Effect of Different M W-EPS on Breast Cancer Cells

The exopolysaccharides samples were tested for their ability to inhibit the proliferation of the 4T1 murin breast carcinoma cells. The antiproliferative activity was determined by the XTT test as described in the Cell Proliferation Kit II (Sigma, 11465015001 Roche, St. Quentin Fallavier, France).

The results were expressed as a percentage of viability via the following formula:

$$\% \text{ Viability} = (\text{A}\_{\text{test}} / \text{A}\_{\text{control}}) \times 100 \tag{3}$$

AControl: Absorbance of untreated cells at 450 nm.

## *4.5. Statistical Analyses*

Data were collected from three independent experiments assayed in triplicate and expressed as the mean ± standard deviation (SD). The statistical significance was analyzed by one-way ANOVA with STATISTICA. 5 software. The post-hoc test of Duncan multiple range was used to perform comparisons. Results were considered as statistically significant with a *p* < 0.05.
