**3. Discussion**

EPS from red microalgae showed several biological activities [28–33] that were generally linked to their molecular weight, rheological behavior [23,34] and their content in sulfate groups and uronic acids. In our study, we evaluated the global and monosaccharide composition, the FTIR footprint, the amount of uronic acids and sulfate groups and the molecular masses of our samples. These results are in accordance with other studies on *P. marinum* EPS [6,7]. EPS from microalgae are complex molecules (much more complex than polysaccharides from other sources), making their complete characterization really challenging. Only a few numbers of EPS coming from microalgae have been studied for glycosidic linkages and none of these studies have led to a complete and defined structure [2]. The cause of this poor knowledge of EPS structures is linked to the fact that these heteropolymers often contains 5 to 10 different monosaccharides, numerous non-sugar substituents such as sulfate, methyl, acetyl and/or pyruvyl groups, and they apparently lack a repeating unit. In a drug development approach, it is mandatory to have fully characterized molecules at the molecular level, including resolution of the glycosidic linkages. However, methods that are classically applied successfully to polysaccharides from other sources (NMR, MALDI-TOF, GC-MS/EI) actually fail to resolve the structure of EPS from microalgae [2]. Even if the elucidation of this complete structure is still lacking, we intended to highlight a potential relationship between structure, physico-chemical properties and activities.

Many researchers have reported that minor changes in molar masses, or viscosity can have positive effects on antitumor activity. In this study, we demonstrate that EPS-2C and EPS-5C show an anti-proliferative activity against breast cancer cells at a high concentration of 2 mg/mL (Figure 6). This activity could be attributed to their lower molar masses and therefore to their low viscosity, thereby increasing their absorption and permeation ability. By further decreasing the molar masses of EPS from *P. marinum*, it could then be expected to increase this anti-proliferative effect. In 2009, Gardeva et al. [34] reported the anti-tumor

activity of *Porphyridum cruentum* exopolysaccharide. They discovered that this sulfated polymer strongly inhibited proliferation of Graffi myeloid tumors in vitro and in vivo. These authors hypothesized that the anticancer activity of the exopolysaccharide extracted from *Porphyridium cruentum* could be associated with its immunostimulatory action as well as its direct cytotoxic properties. In 2012 [35], the anti-tumor and immunomodulatory activities of *P. cruentum* exopolysaccharides of different molar masses were evaluated on the mouse model carrying the S180 tumor in vivo and on the activation of peritoneal macrophages in vitro. The degraded EPSs showed a net immunomodulation dependent on their molar masses as well as the administered concentration. The smallest fragment of 6.53 kDa had the strongest immuno-reinforcing activity, which was in agreemen<sup>t</sup> with the results obtained in our study. Other studies have confirmed that many types of polysaccharides, such as lentinan, *Spirulina maxima* polysaccharide and *Chondrus ocellatus* λcarrageenan, exhibited significant antitumor activity for low molar masses samples [36–38]. In general, the antitumor effects of polysaccharides involve several mechanisms such as modification of the biochemical character of the cell membrane, induction of differentiation and apoptosis of tumor cells and regulation of cell signaling pathways. Nevertheless, immunomodulation is generally considered to be the most important mechanism [39]. It will then be interesting to test lower molar masses samples for antitumoral activity, and to evaluate the immunomodulatory effect to go further in the comprehension of the mechanism.

Bacteria are very common microorganisms in the pathogenic state. The best known and encountered bacteria in the medical sector is *Staphylococcus aureus* which is involved in nosocomial infections [40], but which can also be responsible for food poisoning just like *Escherichia coli* and *Salmonella* [41,42]. However, some bacteria also have developed an increased resistance to clinical antibiotics like methicillin, rendering it ineffective [43]. *Candida albicans* is the most common microorganism among fungal infections, causing high mortality and morbidity especially in immunocompromised patients [44]. The formation of the biofilm represents the virulent form of the pathogen contributing to the pathogenesis of candidiasis. The formation of the *Candida albicans* biofilm is favored by the passage from the unicellular yeas<sup>t</sup> form (blastopore) to the mycelial form (hyphe and pseudohyphe) [45,46], with the ability to strongly adhere to biological surfaces or inert surfaces of medical devices [47]. Biofilm formation leads to high levels of resistance to most conventional antifungal agents, mainly fluconazole and amphotericin B, which have several limitations in terms of efficacy, toxicity, drug interaction and high cost. Thus, the search for alternative strategies and the development of new and more effective antifungal and antibacterial agents is necessary for better therapeutic management. For these reasons, EPS fractions from *P. marinum* were also tested for their antimicrobial and antibiofilm activities. It was found that the native EPS as well as the depolymerized EPS (EPS-2C and EPS-5C) provided an antibacterial and antibiofilm activity with different efficiency levels (Table 3, Figure 5). Our EPS can thus be considered as a powerful inhibitor of bacterial multiplication compared to other exopolysaccharides extracted from other microalgae. De Jesus Raposo et al. [48] indicated that the EPS produced by *Porphyridium cruentum* exhibited antibacterial activity against Gram (+) (*S. aureus*) and Gram ( −) bacteria (*E. coli* and *S.* Enteritidis). Indeed at 1% (*w*/*v*), it clearly inhibited *S.* Enteritidis, reducing CFU to 19%. However, the concentration used in the present study was much lower. The most active fraction was the native EPS at low concentrations that were not showing high viscosity (31.3 μg/mL for the inhibition of the biofilm of *Candida albicans* 110231, 62.5 μg/mL for *anti-Escherichia coli* ATCC 25922 multiplication and 125 μg/mL for anti-*Salmonella* Enteritidis ATCC 13076 and anti-*Staphylococcus aureus* ATCC 29213 growth). Very few polysaccharides from microalgae were described as negatively impacting biofilm formation. The only available study has shown that the cell-bound polysaccharide from *Navicula phyllepta* specifically inhibited biofilm formation from the bacteria *Flavobacterium* sp., while it stimulated biofilm development by *Roseobacter*, and *Shewanella* genera [49]. In comparison with the literature, the efficiency of EPS-0C to inhibit biofilm formation by *C. albicans* observed in this study was greater

(>90%), and this for much lower applied doses (31.3 μg/mL). However, the active dose of EPS-2C and 5C was found to be greater than for EPS-0C, showing that decreasing the molar mass has a negative impact on their efficiency.

The native EPS was also active against *Staphylococcus aureus* methicillin resistant at a concentration equal to 1000 μg/mL (Table 3) showing a significant viscosity (Figure 2A). Therefore, in addition to the functional groups (sulfate and uronic acids, see Table 2) present on EPS-0C, the role of viscosity cannot be excluded from its inhibiting properties on the multiplication of SMR. The rheological behavior of the EPS-2C and EPS-5C fractions was always the same regardless of the applied concentration (2500 μg/mL, 1000 μg/mL or 125 μg/mL) (Figure 2B,C). At these concentrations, these samples have a low viscosity and exhibit a Newtonian behavior. Therefore, the biological activity of these EPSs against bacterial strains and the inhibition of *Candida albicans* biofilm formation could be attributed to their chemical composition, probably primarily to the sulfate and uronic acids groups, as several studies have relied on biological activity with the presence of sulfate groups [33,48,50,51]. Nevertheless, the sulfation degree may not be the only parameter to be considered to explain the biological activity of polysaccharides. Their position and therefore their "accessibility" can improve or not improve the biological activity of the EPS, as demonstrated by [6]. Moreover, several different mechanisms may exist, as non-sulfated polysaccharides (such as chitosan) have exhibited significant efficiency as an antibacterial and antibiofilm agent. For example, Costa et al. [52] have shown that the highest percentage inhibition of biofilm formation by *C. albicans* (66.94%) was obtained for high molar masses chitosan at 0.5 mg/mL and the lowest inhibition percentage (37.97%) was obtained for the low molar masses at 0.75 mg/mL, which was in agreemen<sup>t</sup> with our results. Cobrado et al. [53] also showed that low molar masses chitosan (107 kDa) was able to reduce *C. albicans* biofilm formation up to 41% at a 2.5 mg/mL concentration. For antibacterial activities, results are sometimes controversial, with depending on studies, greater efficiency on Gram (+) bacteria [54,55] or on Gram ( −) ones [56,57]. In fact, the antimicrobial activities of chitosan would be greatly dependent on its physical characteristics, most notably weight average molar masses (Mw) and degree of deacetylation (DD) (for a review, see [58]). Therefore, the antimicrobial mechanisms and structure-function relationships of polysaccharides remain to be elucidated.

#### **4. Materials and Methods**

#### *4.1. Extraction of Exopolysaccharides*

The exopolysaccharide was recovered from a culture of red microalga *Porphyridium marinum*, obtained from Culture Collection of Algae and Protozoa (http://www.ccap.ac. uk/). Cultivation was conducted in 2 L Erlenmeyer flask, containing 1 L of Pm medium [7], under continuous light at 150 μmole photons/m2/s, stirring at 110 rpm and at a temperature of 20 ◦C. The medium was inoculated with 100 mL of a subculture. As previously described [7], synthesis and accumulation of EPS occurred after nitrogen deprivation and entry in a stationary phase. Culture was stopped after 30 days, centrifugated (10,000× *g*, 30 min, 20 ◦C), and the supernatant was desalted and concentrated using a tangential filtration cassette (Vivaflow 200, Sartorius, Göttingen, Germany) having a cutoff threshold of 100 kDa [7]. Finally, the resulting EPS solution was lyophilized before storage at room temperature.

#### *4.2. Preparation of Different-MW Exopolysaccharides*

The exopolysaccharide of *P. marinum* was treated using a High Pressure Homogenizer (HPH, 2.7 kbars, TS HAIVA, Constant Systems LTD, Daventry, UK) to obtain polysaccharide fractions of different molar masses. A polysaccharide stock solution was prepared at a concentration of 2 g/L. Up to five successive cycles were used to obtain 3 samples which were subsequently recovered and lyophilized. The corresponding degraded samples are labeled as follows, according to the number of cycles in the HPH:

