## 3 Results
### Characterization of ED-F1
#### 3.6.2 Fourier Transform Infrared Spectra Analysis  
ED-F1 was subjected to FT-IR spectroscopy to determine the characteristic absorption bands and _i_-carrageenan was used as a standard. In both samples, two similar bands were observed in the 4000-2000 cm\({}^{-1}\) region of the FT-IR spectra (Figure 6). The broad absorption appeared at 3200-3500 cm\({}^{-1}\) represents hydrogen bonded O-H stretching vibrations while a weak signal at 2926 cm\({}^{-1}\) is due to C-H stretching vibrations. The FT-IR absorption band observed around the region of 1075-1041 cm\({}^{-1}\) was attributed to glycosidic linkages connecting sugar molecules and a common trait to all polysaccharides  
Figure 5: _E. denticulatum_ ED-F1 fraction observed under size-exclusion chromatography (HP-SEC).  
\begin{table}
\begin{tabular}{c c c c c c} \hline \multicolumn{2}{c}{**Optimum Conditions 1**} & \multicolumn{2}{c}{**Responses 2**} & \multicolumn{2}{c}{**Desirability = 0.844**} \\ \hline Temperature (\({}^{\circ}\)C) (In range) & Time (Min) (In range) & H\({}_{2}\)SO\({}_{4}\) (M) (In range) & Predicted & Actual &
\begin{tabular}{c} \% Relative \\ error \\ \end{tabular} \\ \hline
121 & 21 & 0.12 & \
\begin{tabular}{c} ED-F1 Yield (g/100 g) \\ (Maximize) \\ HMF (g/L) \\ (Minimize) \\ \end{tabular} & 10.81 & 11.15 \(\pm\)0.03 & 3.15 \\ \hline \end{tabular}
\begin{tabular}{c} 1 X\({}_{1}\) Temperature: hydrolysis temperature; X\({}_{2}\) Time: hydrolysis time and X\({}_{3}\) H\({}_{2}\)SO\({}_{4}\): sulfuric acid concentration. \\ 2 ED-F1: _E. denticulatum_ fraction 1; HMF: 5-hydroxymethyl furfural.  
\end{table}
Table 3: Validation of conditions for the optimum yield of ED-F1.  
and oligosaccharidesacharides. The band around 930 cm\({}^{-1}\) were ascribed to the vibration of C-O-C bridge of 3,6-anhydrodialacose. These bands were also observed in earlier studies in both agaro-oligosaccharides from enzymatic hydrolysis of agar [61], sulfated polysaccharides extracted from red seaweeds [62]. The 3,6-anhydrogalactose polymer unit is one of the unique features that distinguished agaran and carrageenan type oligosaccharidesacharides from other common seaweed derived polymer such as alginate type oligosaccharidesacharide [63; 64]. However, the 930 cm\({}^{-1}\) peak is known to be absent in both _mu_ and _lambda_ carrageenanan as the C-O-C bridge is replaced by a sulfate group RO-SO\({}_{3}\)[64].  
Sulfation observed in ED-F1 was similar to the \(i\)-carrageenanan standard designated at C4 of the galactose unit (841 cm\({}^{-1}\)) and C2 of the 3,6-anhydrodialacose (805 cm\({}^{-1}\)) [63]. A small peak near 1370 cm\({}^{-1}\) also indicates the presence of sulfate groups in both samples. A broad IR peak was also observed between 1120 and 1270 cm\({}^{-1}\) and is indicative of the S = O stretching vibration of sulfate groups on both sample [62]. A slightly higher intensity of 890 cm\({}^{-1}\) peak was observed in ED-F1 but almost unobservable at \(i\)-carrageenanan. This peak represents the stretching vibration of the anomeric C-H of unsulfated \(\beta\)-galactopyranosyl residues implying the presence of this residue in ED-F1 but very minimal in \(i\)-carrageenan. A previous study by [60] compared the prebiotic activity of sulfated and non-sulfated seaweed derived oligosaccharidesacharides namely alginate oligosaccharidesacharide, agaro- oligosaccharidesacharide and \(k\)-carrageenan oligosaccharides. Sulfated and non-sulfated oligosaccharides have been shown to have distinct effects on the gut microbiota in terms of the types of beneficial bacteria groups promoted, opportunistic pathogen population and production of different ratios of short chain fatty acids (SCFAs). Thus, the prebiotic efficacy of ED-F1 having both sulfated and non-sulfated \(\beta\)-galactopyranosyl residues in comparison with other either sulfated or non-sulfated marine oligosaccharides need further verification.