Extraction and characterisation of sodium alginate from the Southern African seaweed Ecklonia maxima

2.4.1 Protein content determination

The Bradford assay was used to determine the contaminant protein content of the sodium alginate extracts, with bovine serum albumin as a suitable standard (Bradford 1976).

2.4.2 Phenolic content determination

The total phenolic content in the extracts was determined as described by Malgas et al. (2016) using the Folin-Ciocalteu method. Gallic acid was used as a suitable standard, and the phenolic content was expressed as % of gallic acid equivalents per g dry weight.

2.4.3 Total sugar determination

The total sugar content in the extracted sodium alginate was determined by performing the phenol-sulfuric acid method described by DuBois et al. (1956). The samples were made to 1 mg/mL using dH₂O, and 100 µL of this sample was added to 300 µL of concentrated sulfuric acid, followed by 50 µL of 5 % phenol (w/v). The samples were mixed, incubated at 90 °C for 10 min, and then cooled to room temperature. A volume of 200 µL of the sample was then transferred to a 96-well microtiter plate, and the absorbance readings were taken at 490 nm using a PowerWaveX™ spectrophotometer with KC Junior software^® (Winooski, Vermont, USA). The total sugar content of sodium alginate was calculated from the phenol-sulfuric standard curve constructed using varying concentrations of commercial sodium alginate (0–1 mg/mL).

2.4.4 Total uronic acid determination

The total uronic acid content of the sodium alginate was determined by performing the 96-well carbazole assay method described by Cesaretti et al. (2003). Varying concentrations of sodium alginate samples (0–1 mg/mL) of 50 µL were placed in a 96-well plate. Sodium tetraborate in sulfuric acid, 200 µL, with a concentration of 25 mM, was added into the wells, followed by heating the plate at 100 °C for 15 min in an oven. A volume of 50 µL 0.125 % carbazole in absolute ethanol was added and then heated at 100 °C for 10 min in an oven. The plate was cooled for 15 min at room temperature and read using a PowerWaveX™ spectrophotometer with KC Junior software^® (Winooski, Vermont, USA) at 550 nm. The total uronic acid content of sodium alginate was calculated from the mannuronic acid standard curve constructed using varying concentrations (0.1–1 mg/mL).

2.5.1 Fourier-transform infrared (FTIR) spectroscopic analysis

FTIR analysis was used to characterise the sodium alginate samples using a Spectrum 100 spectrometer system (Perkin Elmer, Wellesley, MA, USA) with Spectrum™ One software. Baseline and ATR corrections for penetration depth and frequency variations were carried out using this program. The samples were pressed uniformly and tightly against the spring-loaded anvil, and the spectra were recorded from 4,000 to 600 cm⁻¹.

2.5.2 Nuclear magnetic resonance (NMR) spectroscopic analysis

The sodium alginate extracts (1 mg/mL) were dissolved in D₂O, followed by mixing. The deuterium-exchanged samples were subjected to ¹H-NMR analysis. Spectra were recorded at 70 °C using a Bruker 400 MHz spectrometer (Billerica, Massachusetts, USA) with Mnova 15.0.1 software (Mestrelab Research, Santiago de Compostela, A Coruña, Spain). The chemical shifts were expressed in ppm relative to tetramethylsilane (TMS) or the residual solvent signals.

2.5.3 Circular dichroism (CD) spectroscopy analysis

A sodium alginate solution of 0.25 mg/mL was scanned using a Chirascan spectrophotometer (Applied Photophysics Limited, Leatherhead, South East, United Kingdom) three times at 80 nm/min with 1 nm slit width and a time constant of 1 s. Data were collected from 190 to 350 nm at 1 nm intervals. The M/G ratio was calculated by the parameters described by Morris et al. (1980): mannuronate/guluronate ≈ 2.0(peak/trough), if peak/trough < 1 or % mannuronate ≈ 27(peak/trough) + 40, if peak/trough >1.

2.5.4 Determination of the intrinsic viscosity and molecular weight

A 10 mg/mL sodium alginate stock solution was prepared in 100 mM saline water, and 0.1–10 mg/mL dilutions of the stock solution were prepared. After vortexing the solution for 5 s, 1 mL of each was loaded on a semi-micro viscometer (Cannon Instrument Company, State College, PA, USA). The flow time of the solution from the top to the bottom meniscus was measured. The intrinsic viscosity was calculated by using the formulae:

where η_r is the relative viscosity, η is the sodium alginate solution viscosity (mPa s), η_s is the solvent viscosity, [η] is the intrinsic viscosity (L/g), and C is the sodium alginate concentration (g/L).

The following formula estimated the molecular weight of sodium alginate by intrinsic viscosity, where k and a are sodium alginate-specific constants (Halabalová et al. 2004).

The parameters of k and a were substituted with the Mark–Houwink–Sakurada (MHS) parameters of Vold et al. (2006):

when I = 0.1 M,

where [η] is mL/g and M_w is g/mol.

2.5.5 Congo red polysaccharide folding analysis

A modified Congo red assay described by Yang et al. (2021) was performed. A 2 mg/mL concentration of sodium alginate was prepared. Congo red was prepared to 80 μmol/L, which was dissolved in different concentrations of NaOH ranging from 0 to 0.5 mol/L (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mol/L) to be mixed in a volume ratio of 1:2. The mixture was incubated for 1 h at 25 °C. Distilled water was used for the control. The total absorbance was scanned from 450 to 510 nm using a PowerWaveX™ spectrophotometer with KC Junior software.

2.5.6 X-ray powder diffraction analysis

The crystallinity of the sodium alginate was measured using a Bruker D2 2nd Gen Phaser (Billerica, Massachusetts, USA). Samples were scanned from 2θ (Theta) of 5–60° with a step size of 0.02° per second. The relative crystallinity of the polysaccharide was calculated by dividing the area of the diffraction peak at 2θ by the total area of 17–21°.

2.5.7 Thermogravimetric analysis of sodium alginate samples

Commercial sodium alginate and sodium alginate extracted from E. maxima samples were subjected to thermogravimetric analysis (TGA). TGA was conducted with a PerkinElmer^® (Pyris Diamond model) thermogravimetric analyser (Waltham, USA) following the method of Mabate et al. (2021). The samples were placed in an alumina crucible and heated from 30-700 °C with a heating rate of 10 °C min⁻¹ whilst continuously flushing the apparatus with argon gas at a 60 mL/min atmospheric pressure flow rate.

3.2.1 Functional group determination of sodium alginates by FTIR spectroscopy

FTIR spectra in the 4,000–600 cm⁻¹ range were collected for the commercial and extracted sodium alginates (Figure 1). Typical sodium alginate spectra were shown for both sodium alginates. Extracted sodium alginate showed all the typical polysaccharide bands of O–H stretching vibrations at 3,300 cm⁻¹, asymmetric stretching of carboxylate O–C–O vibration at 1,600 cm⁻¹, and symmetric stretching vibration of the carboxylate group at 1,400 cm⁻¹ (Fertah et al. 2014; Silverstein et al. 1991). The absorption bands in the 950 cm⁻¹, 900 cm⁻¹, and 810 cm⁻¹ regions indicated the C–O stretching vibration of uronic acid residues, Cl–H deformation vibration of β-mannuronic acid residues, and characteristics of mannuronic acid residues, respectively (Belattmania et al. 2020; Fertah et al. 2014). Peaks at 1,230–1,280 cm⁻¹ (attributable to sulphated polysaccharides such as fucoidan) were absent, indicating highly purified sodium alginate (Rashedy et al. 2021). In addition, there were no peaks in the 1755 cm⁻¹ region (an indication of stretching of the protonated carboxylic group). Instead, the peaks appeared at 1,600 cm⁻¹, indicating asymmetric and symmetric stretching vibration of the free carboxyl group of sodium alginate, which showed the displacement of the proton with a sodium ion (Taha et al. 2005). FTIR analysis was not able to identify the presence of guluronic acid residues. NMR was subsequently used to confirm the presence of mannuronic and guluronic acid residues in the sodium alginate extracts.

Figure 1:

Fourier-transform infrared spectra of sodium alginate samples. The red line represents the commercial sodium alginate, and the blue line represents the sodium alginate extracted from Ecklonia maxima using the optimised method.

3.2.2 Linkage analysis of the sodium alginates by ¹H NMR

The three key signals, the anomeric proton of guluronic acid at 5.1–5.2 ppm, the anomeric protons of mannuronic acid, the H-5 of alternating blocks at 4.7–4.9 ppm, and the homopolymeric G blocks H-5 of guluronic acid residues at 4.5–4.6 ppm, are frequently used in the literature to identify and report on the components in sodium alginates (Belattmania et al. 2020; Fertah et al. 2014). The three key signals for sodium alginates were present in the ¹H-NMR spectra generated from the E. maxima extracts and the commercial sodium alginate samples (Figure 2). However, the solvent peak of D₂O was shown around the 4.75 ppm region, which overlapped with the mannuronic acid protons and the H-5 of alternating blocks. Although running the NMR at a temperature of 70 °C was used to overcome this problem, the sodium alginate peaks shifted with the solvent peak. However, the maximum intensity at 4.7–4.9 ppm still revealed the presence of a D₂O solvent peak for both the commercial sodium alginate and the extracted sodium alginate. On the other hand, both sodium alginates showed peaks at 4.5–4.6 ppm, indicating the presence of homopolymeric guluronic blocks. A peak at around 5.1–5.2 ppm was also observed for both the commercial and extracted sodium alginates, indicating the presence of guluronic acid units, with extracted sodium alginate with higher intensity than the commercial sodium alginate. Although the NMR revealed the building blocks of commercial and extracted sodium alginate, the M/G ratio could not be identified due to the high solvent peak. Therefore, CD spectroscopy was used to identify the M/G ratios of the sodium alginates.

Figure 2:

Nuclear magnetic resonance spectra of sodium alginate samples. The red line represents the commercial sodium alginate, and the blue line represents the sodium alginate extracted from Ecklonia maxima using the optimized method.

3.2.3 Characterisation of the sodium alginate M/G ratio by CD spectroscopy

CD spectroscopy was performed to determine the conformational changes in the polysaccharides’ secondary and local tertiary structures and to determine the mannuronic and guluronic ratios. Carbohydrates do not form defined structures like the α-helices and β-sheets in proteins, but they may be in the form of a disordered chain, an extended rigid chain, or a collapsed, flexible and helix-like chain (Sun et al. 2018). As shown in Figure 3, sodium alginate exhibited positive and negative Cotton effects at 199 and 213 nm, respectively, indicating the presence of helix-like and sheet-like structures (Sun et al. 2018). However, the peaks at 199 nm and the troughs around the 213 nm region displayed different values between the commercial and extracted sodium alginate samples, indicating different structural conformations. The mannuronate and guluronate ratios of the commercial and extracted sodium alginate were determined by a parameter described by Morris et al. (1980). The commercial sodium alginate peaked at around −5.63, and the trough was around −9.08. Using the first parameter, the M/G ratio of the commercial sodium alginate revealed 43 % mannuronate and 57 % guluronate content. The extracted sodium alginate peaked at around 0.48, and the trough was around −8.49; therefore, the M/G ratio was 1.86, indicating 65 % mannuronate and 35 % guluronate content. The M/G ratios of commercial and extracted sodium alginate were 0.76 and 1.89, respectively. Although these values did not correspond to the chemical profiles reported for a typical sodium alginate, Saji et al. (2022), also using CD spectra, reported M/G ratios from different seaweed species in the range of 0.43–2.52.

Figure 3:

CD analysis of commercial sodium alginate samples. The red line represents the commercial sodium alginate, and the blue line represents the sodium alginate extracted from Ecklonia maxima using the optimized method.

M/G ratios obtained for different species of brown seaweeds and different commercial sodium alginates varied (Table 2). The results of our study showed that the commercial sodium alginate contained comparatively less mannuronic acid; the E. maxima extracted sodium alginate contained more mannuronic acid, but less compared to what has been reported in other commercial sodium alginates (Belattmania et al. 2020), or brown seaweeds (Rashedy et al. 2021), 15.58 % and 30.11 % less, respectively. Our results similarly showed that the commercial sodium alginate had a comparatively low M/G ratio; the E. maxima extracted sodium alginate had a higher M/G ratio, but much less compared to what has been reported in other commercial sodium alginates (Belattmania et al. 2020), or brown seaweeds (Rashedy et al. 2021), 77.78 % and 94.14 % less, respectively. The M/G ratio of alginate has been found to affect its gel strength and permeability. A high M/G ratio results in a weaker structure but a more permeable gel matrix, while a low M/G ratio leads to a stronger structure that is less permeable (Khanna et al. 2010; Ramos et al. 2018). Thus, the desired M/G ratio ranges intermediately between permeable and less permeable, which balances permeability and strength for general use. However, it should be dependent on the application of the sodium alginate gel. Extracting sodium alginate from an endemic South African brown seaweed to produce a desirable biopolymer with both permeable and strong gel characteristics could be explored. The endemic brown seaweed, Ecklonia maxima, may prove to be a promising candidate.

3.2.4 Molecular weight and intrinsic viscosity of the sodium alginates

The viscosity average molecular weights of the commercial and extracted sodium alginates were calculated from the Mark-Houwink-Sakurada (MHS) equation following the method of Vold et al. (2006). Their intrinsic viscosities were calculated by equations (1)–(3). The average intrinsic viscosity of the commercial sodium alginate was 5,728.19 mL/g, and for the extracted sodium alginate, 7,307.02 mL/g. The molecular weights of the commercial and extracted sodium alginates were calculated according to equations (4) and (5) with the MHS parameters of Vold et al. (2006). Using these equations, the average molecular weight of the commercial sodium alginate was calculated to be 326 kDa, while that of the extracted sodium alginate was 429 kDa. These molecular weights were higher compared to those of another brown seaweed, Laminaria digitata, reported by Vauchel et al. (2008, 105 kDa) and Fertah et al. (2014, 114 kDa). The extracted sodium alginate was shown to have a more comparable molecular weight to E. radiata from South Australia, which showed a molecular weight range of 373–986 kDa (Lorbeer et al. 2015).

3.2.5 Characterisation of sodium alginate structure by Congo red

A Congo red test determined whether the commercial and extracted sodium alginates exhibit a triple helix conformation. Congo red can form complexes with the helical conformation of polysaccharides (Guo et al. 2018). The maximum absorption wavelength λ_max of polysaccharides will generate a bathochromic shift in the wavelength range of 400–600 nm, as opposed to no bathochromic shift for pure Congo red (Guo et al. 2018). The commercial and extracted sodium alginates showed a bathochromic shift with Congo red compared to pure Congo red (Figure 4). This phenomenon suggested that the commercial and extracted sodium alginates are in a triple-helix structure form (Yang et al. 2021). It has also been reported that the λ_max of the polysaccharide complex with helical conformation can also show a change to a longer wavelength at low concentrations of NaOH and decrease with increasing NaOH concentration (Guo et al. 2018).

Figure 4:

Congo red spectroscopy analysis of the sodium alginate samples. The purple line represents the Congo red standard, the red line represents the commercial sodium alginate, and the blue line represents the sodium alginate from the extracted Ecklonia maxima using the optimized method.

3.2.6 Characterisation of sodium alginate morphology by X-ray powder diffraction

X-ray powder diffraction is a technique widely used to determine the structure of different substances. Repeating unit polysaccharides can be arranged in an orderly structure within their molecule structure and thus give a certain diffraction pattern when irradiated with X-rays on their surface (Qian et al. 2009; Wang et al. 2014). Usually, polysaccharides lead to ‘bun-shaped’ XRD curves due to poor crystallisation capacity (Wang et al. 2014). The XRD spectra of the commercial and extracted sodium alginates exhibited ‘bun-shaped’ diffraction peaks when 2θ (2-Theta) was about 17–20° (Figure 5), indicating that both the commercial and extracted sodium alginates had an amorphous structural makeup; this has been reported for sodium alginate before (Helmiyati and Aprilliza 2017).

Figure 5:

X-ray diffraction analysis of the sodium alginate samples. The red line represents the commercial sodium alginate, and the blue line represents the sodium alginate extracted from Ecklonia maxima using the optimized method.

3.2.7 Thermogravimetric analysis of the sodium alginates

Thermogravimetric (TGA) analysis and derivative thermogravimetry (DTG) analysis showed that both commercial and extracted sodium alginate samples lost weight slightly at approximately 50–250 °C (Figure 6A). The weight loss could be due to moisture loss and volatile compounds in the samples (Gufe et al. 2023). The TGA analysis showed greater weight loss between 250 and 300 °C and slower weight loss from 300 to 750 °C. The DTG analysis concurred with the TGA curve, showing a similar exothermal peak at 250–300 °C for the commercial and extracted samples (Figure 6B), reaching 50 % of their original weight. This was attributed to sample degradation, resulting in the formation of water and CH₄ and the release of CO₂ (Salisu et al. 2015). The last weight loss happened at around 300–750 °C, to approximately 20 % and 30 % of the ash content for the commercial and the extracted sodium alginates, respectively (Table 1). The difference between the commercial and the extracted sodium alginate could be due to the difference in the inorganic compounds or the sodium carbonate remaining in the E. maxima samples after extraction. However, the E. maxima samples showed a typical sodium alginate TGA profile and only contained a few contaminants compared to the commercial sodium alginate.

Figure 6:

Thermal decomposition characteristics of sodium alginates. (A) Thermogravimetric analysis and (B) derivative thermogravimetry analysis of the sodium alginate samples. The red line represents the commercial sodium alginate, and the blue line represents the sodium alginate extracted from Ecklonia maxima using the optimized method.