# Marine microalgae Biomolecules and Their Adhesion Capacity to _Salmonella enterica_ sv. Typhimurium
## 3 Results
### Extraction and Characterization of Polysaccharides from P. gyrans and P. lutheri
The \({}^{13}\)C NMR spectra of GW-p (Figure 3a) and LW-p (data not shown) demonstrated six major signals at 102.4, 72.3, 85.6, 67.9, 75.8, and 60.4 ppm, which were assigned to the six-membered ring of \(\beta\)-d-Glc\(p\) units (residue G\({}_{\mathrm{L}}\)), the major component of these fractions. The anomeric signal at 4.53 ppm (\(J_{\mathrm{H1,H2}}\) 7.7 Hz) in the \({}^{1}\)H NMR spectrum confirmed the \(\beta\)-anomericity of these units. The cross-peak in \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Sugar Derivative 1**} & \multirow{2}{*}{**Deduced Linkages 2**} & \multicolumn{2}{c}{**Molar Ratio 3**} \\ \cline{3-4}  & & **GW-p** & **GW-s** \\ \hline
2,3,4,6-Me\({}_{4}\)-Glc 1  & Gl\(p\)-(1\(\rightarrow\) & 7.3 & 38.0 \\
2,4,6-Me\({}_{3}\)-Glc 1  & 90.0 & 26.7 \\
2,3,4-Me\({}_{3}\)-Glc 1  & \(\rightarrow\)6)-Glc\(p\)-(1\(\rightarrow\) & 2.5 & 11.2 \\
2,4-Me\({}_{2}\)-Glc 1  & 5.3 & 24.1 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Methylation analysis of GW-p and GW-s.  
the HSQC spectrum (Figure 4a) at 3.49/85.6 ppm indicated the 3-O-substitution of \(\beta\)-d-Glc\(p\) residues. The non-substitution at O-6 was confirmed by the \({}^{13}\)C-DEPT inverted resonance at 60.4 ppm. All the \({}^{1}\)H and \({}^{13}\)C NMR assignments of GW-p are shown in Table 4. The spectroscopic analyses indicated the occurrence of a (1\(\rightarrow\)3)-linked \(\beta\)-glucan in the GW-p fraction. The \({}^{13}\)C NMR analyses of fractions GW-s (Figure 3b) and LW-s (data not shown) showed signals of (1\(\rightarrow\)3)-linked \(\beta\)-Glc\(p\) residues at 102.4, 72.2, 85.6, 67.9, 75.8, and 60.4 ppm, which were attributed to C-1-C-6, respectively. Additionally, the HSQC spectrum (Figure 4b) contained anomeric correlations (H-1/C-1) at 4.38/103.5 and 4.23/102.5 ppm, corresponding to non-reducing end units (residues G\({}_{3t}\) and G\({}_{6t}\), respectively), and others at 4.28/102.6 and 4.36/102.4 ppm, corresponding to 6-(G\({}_{\rm B}\)) and 3,6-di-\(O\)-substituted Glc\(p\) residues (G\({}_{\rm C}\)), respectively. Furthermore, the HSQC cross-peaks at 5.00/91.5 and 4.39/96.1 ppm were attributed to H-1/C-1 of \(\alpha\)- (G\({}_{\alpha}\)) and \(\beta\)-reducing end Glc\(p\) residues (G\({}_{\rm B}\)), respectively. The correlation at 3.40/87.0 ppm arose from a substitution at O-3 in (1\(\rightarrow\)3,6)-linked \(\beta\)-d-Glc\(p\) units, while those at 3.23/75.6, 3.24/75.6, and 3.23/75.8 ppm were from free O-3 of non-reducing ends and (1\(\rightarrow\)6)-linked \(\beta\)-d-Glc\(p\) units. The \({}^{13}\)C DEPT-135 inverted resonance at 67.9 ppm corresponded to substituted CH\({}_{2}\)-6 of \(\beta\)-d-Glc\(p\) units, while the inverted signals at 60.7 and 60.4 ppm, could be attributed to unsubstituted CH\({}_{2}\)-6 of \(\beta\)-d-Glc\(p\) units. All the \({}^{1}\)H and \({}^{13}\)C NMR assignments, based on HSQC (Figure 4), COSY (Figure S1), and TOCSY (Figure S2) spectra, presented in Table 4 are in agreement with previously reported resonances [38; 39; 40; 41]. In accordance with the methylation analysis, the NMR data suggest the presence of a highly branched \(\beta\)-d-glucan constituted by a (1\(\rightarrow\)3)-linked main chain substituted at O-6 by single units of D-Glc\(p\) or (1\(\rightarrow\)6)-linked \(\beta\)-d-Glc\(p\) branches. The HSQC NMR spectrum (Figure 5a) of the alkaline fraction LA-p, showed anomeric correlation at 4.59/98.4 ppm (\(J_{\rm H1,H2}\) 3.7 Hz), which is consistent with an \(\alpha\)-glycosidic configuration. Correlations at an unusual high field region (2.89;2.64/54.9 ppm) indicated the presence of a -CH\({}_{2}\)-SO\({}_{3}\)\({}^{-}\) group of 6-_sulfoquinovose_ units. The \({}^{13}\)C-DEPT inverted signal at 54.9 ppm (CH\({}_{2}\)) confirmed the S-substituted C-6 (Figure 5c'). The H-2/C-2-H-5/C-5 correlations at 3.22/71.3, 3.4/72.6, 3.02/74.3, and 3.82/68.1 ppm, respectively, also confirmed the presence of a quinovosyl structure (residue SQ).