Alginate samples were extracted from the brown algae (Phaeophyceae), a common source of all samples with varying initial Mw. Analytical grade reagents including NaCl, CaCl₂, and NaN₃ were also utilized in this study (purchased from Fisher Scientific, UK).

The irradiation breaks down the Mw and the alginates into smaller units of molecules (M-/G-blocks). Hence, the degradation produces polymers with low Mw, manifesting the critical conditions of low and high doses of γ-radiation. A single dose of 15 kGy was applied to the commercial alginate samples. A correlation of experimental results following the different methods is stated below.

Alginate samples at different concentrations, 10 ml (w/w) in 0.01 M NaCl were prepared to allow a continuous shaker to obtain a clear solution. The pH buffer series (4.0, 7.0, and 9.2) were prepared for calibration of the pH meter, followed by pH measurement using a Benchtop pH meter (Orion 4 Star, pH-ISE, Thermo Electron Corporation).

The changes in the relative viscosity of the prepared alginate solutions were assessed using an Ubbelohde-type (Cannon Ubbelohde Semi-Micro Calib 75) capillary viscometer during binding with 0.01 M NaCl at 25°C ± 1°C (Sariyerli et al., 2018).

The 1 L 0.01 M NaCl solvent was prepared and filtered using a 0.8 microns cellulose acetate membrane (Na-glene, 25 mm) vacuum filter, followed by the addition of 0.005% sodium azide (NaN₃) to protect the bacterial growth. The different concentrations of alginate samples 10 ml (w/w) were dissolved in the solvent and left overnight on a roller to allow continuous shaking for clear solutions. Following homogenization, the time flow (in seconds) of the solvent between the two marks of a calibrated Ubbelohde viscometer was measured. This procedure was also repeated with 2 ml alginate solution. The solution was also filtered using a 0.8 microns cellulose membrane to remove insoluble materials. The time to flow between the two marks of the viscometer was measured twice. If the difference error was less than 2 s, both the readings were averaged, but if the error was more than 2 s, a third measurement was recorded before averaging the time. However, if the readings were significantly different a fresh batch of samples was prepared. If the time recorded for the alginate emulsion was between 2 and 3 times that of the solvent, the sample was diluted, and a longer time recorded required the sample to be diluted again before measurement, whereas a shorter time recorded required the preparation of a more concentrated solution. Next, 0.4 ml of the filtered solvent (0.01 M NaCl) was added to the viscometer to measure the viscosity. The final measurement (time of flow) was repeated by successively adding 0.6, 1, 2, and 4 ml of solvent.

The Gel Permeation Chromatography coupled with a multi-angle laser light scattering (Dawn DSP, Wyatt Technology Corporation) was employed to assess the Mw distribution and polymeric system parameters (Feng et al., 2017; Hadar et al., 2019). The 0.1 M NaCl solvent was prepared and filtered using Cellulose Nitrate membrane (pore size: 0.22 µm, dia: 0.47, Thermo Scientific, DS0200-4020, made in the United States). Approximately 50–100 ml of the solvent was used to rinse the vacuum flask gently before filtering the rest of the amount. Then, 0.005% NaN₃ was added to prevent bacterial growth. The 10 ml (w/w) sample (1 mg/ml concentration) prepared under considering the following moisture content calculation: Exact Sample = sample (g) × (1- moisture content/100). The RI and UV detectors were set to auto-zero before injecting samples into the system. Afterword, 1 mg sample was injected into the system through the filter (membrane 200, MF 1.2) at a pump flow rate of 0.45 ml/min, pressure of the whole system was 1.99–2.2 Mpa, using 11 detectors in UV scattering, light scattering wavelength was 280 nm, 35°C, and 0.2 μW. At the end of the run, the Mw, radii of gyration, and percentage mass recovery (calculated mass/injected mass×100) were recorded.

To determine the Mark-Houwink constant, a plot was plotted taking the logarithm of intrinsic viscosity against the logarithm of Mw (M) obtained from GPC-MALLS, using the equation: [ η ]   =  log k  + α  × log M The slope of a straight line represents the ɑ-value, while the intercept represents the (Mark-Houwink-Equation) k value is l. The values of the constants K and ɑ were 8.1 × 10^–3 ml/g and 0.92, respectively (El-Mohdy, 2017; Masimov et al., 2019).

To measure the solubility, the gel was prepared following the “procedure”, then filtered using an MF 200, size 70 mm (pore size 1.2 mm) glass microfibers and weighted as W1. The weight (W) of the glass fiber membrane was measured after drying it in an oven at 105°C for 1 h, before subsequently chilling it in the desiccator with silica gel. The method that was approved by the Joint Expert Committee on Food Additives (JECFA) for hydrocolloids, was adopted by changing the solvent from mild alkaline to water. To determine the gel fraction, test materials depending on mesh size likely a 20-mesh steel screen (1,041 µm) can be used (H. Gulrez et al., 2011). The gel strength was determined using two steps namely in 50 ml of water and by adding 1 ml of 0.5 M CaCl₂ gelling solution dropwise into 50 ml of water as a binding agent (Artiga-Artigas et al., 2017). Described procedure was followed for 50 ml in water solution and 50 ml in water by adding the gelling agent. The filter paper dried in an oven at 105°C until it cooled and reached a constant weight. The percentage gel strength was calculated using: Percentage Gel Strength = ( W 1 − W ) / W × 100

The % Gel Strength increased = % Gel strength with CaCl₂ - % Gel strength in water.

Step 1: The 0.5% alginate (100 ml) solution was prepared by adding 0.5 g of alginate into 100 ml warm distilled water while stirring (using magnetic flee) continuously for 15 min at 60°C. Subsequently, 0.5 M CaCl₂ (50 ml) solution was prepared and kept separately. A little amount was taken in a universal vial then picked up a 1 ml syringe and kept it in a safe place.

Step 2: Ensured the sample is fully dissolved. If the sample is not fully dissolved, stirring was continued until no visible insoluble materials. Once fully dissolved, 50 ml of the exact solution (0.5% stock) was transferred into a beaker and kept aside.

Step 3: The remaining solution was filtered (while hot) according to the following method. The weight of an MF 200, size 47 µm glass microfiber was determined (W1) before filtering the remaining 0.5% alginate solution. Introduced a small portion of the solution to the filter then started pump for filtering gently. Then put the full sample to be filtered accurately. The beaker should be rinsed with exact 5 ml distilled water and the solution filtered again. The same process was repeated for all samples, with and without gel solution.

Step 4: Following filtrations, the filter paper was gently removed from the vacuum filter system and dried in a dryer overnight (with labels on aluminum foil).

Step 5: A 1 ml volume of 0.5 M CaCl₂ solution was added dropwise into a beaker with the solution from Step 2 while stirring for 5 min. After stirring, Step 3 and Step 4 were repeated with a 1 ml syringe. The filter paper was transferred to aluminum foil and dried overnight at 105°C.

Step 6: The next day, the filter papers from the dryer were immediately transferred into a desiccator before the filter papers were measured. The pre- and post-weight were measured to determine the gel strength following the equation.

The DP is defined as the number of monomers in a macromolecule or polymer or oligomer molecule (Anshuman, 2018). The atomic mass/Mw of the monomeric per unit of alginates samples has taken 200 and calculated the DP followed by DP = Total Mw/atomic weight of the monomeric per unit (200).

The experimental data is made up of the results obtained from the mean averages of three replications of the samples. A sample is taken and run through the system three times, and the mean average is considered for the data presented. The data were analyzed using the M-STAT program, where the covariance was accepted at 0.01 ≥ 0.05 level of significance.

In the irradiated samples, the dissociated monomeric units with small molecules are capable of absorbing water through inter- and intra-molecular pores. Therefore, the moisture content significantly increased for all samples following irradiation. Based on Table 1, the pH values were recorded in the range of 5.2/5.3 (sample 7) to 7.2/7.0 (sample 3) in the control/irradiated sample. Although the pH values were not significantly different in terms of radiation, a small variation was observed among the samples. The pH values indicated a slight dissociation of the carboxylic acid groups. Meanwhile, in an aqueous alginate solution, two types of forces were identified, the repulsive forces among the ionized carboxylic -COO- groups and the H-bonding formed between ionized carboxyl groups and carboxylic acid groups. These two forces are opposite to each other. At a high pH values exceeding the pKa value (3.7) of the uronic acid groups, the mutual repulsive forces between the ionized carboxyl groups because the network structure to loosen. Whereas, at a low pH, the electrostatic interactions are weakened promoting the intermolecular H-bonds leading to possible entanglements (Tally and Atassi, 2015; Rizwan et al., 2017; Ghobashy and Bassioni, 2018; Olad et al., 2018; Shivakumara and Demappa, 2019).

The determination of the viscosity of alginate solutions in water is not suited for characterizing alginate samples (data not presented). The alginate solutions presented electrolytic behavior in a water solvent. There was no significant linear relationship observed between the viscosity and concentration of the alginate aqueous solutions in water. Although the findings are under review, the effects of salts were most pronounced at low concentrations of alginate. When the concentration of alginate was increased, the ionic strength increased. Moreover, at a sufficiently high alginate concentration (approximately 3%), an increase in ionic strength was also observed through the addition of inorganic salt to the solution that produced very little or no effect on the viscosity (Wu et al., 2018). Generally, polyelectrolytes possess intrinsic viscosity that is reciprocally linear to the square root of ionic strength (Figure 1). It is well known that the viscosity of alginates is ionic strength dependent strongly. However, the relationship to the ionic strength has not yet been established (Chen et al., 2019).

To determine the Mv of the alginates, the Mark-Houwink equation was used: [ η ]   =  kMa Where [η] is the intrinsic viscosity, k and a are values of constants, and M is the Mv. A plot of log η vs Mw yielded the α value from the slope of the straight line and log k value from the axis intercept. The control constants α and k were calculated as 0.94 and 7.25 × 10^–5, respectively, while the constants of the irradiated samples were calculated as 0.91 and 8.78 × 10^–5, respectively (Figure 2; Figure 3).

The Mark-Houwink’s parameter α for the control and irradiated samples ranged from 0.91 to 0.94 and from 7.254 × 10^–5 to 8.78 × 10^–5 for K at different ionic strengths of the alginate samples. Polymer or bio-polymer samples with linear chain stature had no branches and yielded α values of 0.6–0.8, whereas branched sampled yielded lower α values, and stiff chain with stretched samples yielded higher values (Mudgil et al., 2018). Therefore, the α values were calculated in this experiment to confirm the open and stiff structure of the samples, because Table 1 listed lower values for the irradiated samples.

The GPC was used to determine the molecular distribution of all samples. Table 1 presents the Mw, percentage mass recovery, radii of gyration (Rz/Rg), and percentage reduction of Mw. The Mw and radii of gyration decreased due to the effects of γ-radiation. The Mv of intrinsic viscosity is usually higher than the Mw. The viscosity average molecular weight depends on intrinsic viscosity which is reliant on ionic strength of the solution and the interaction between per unit helical residues, and it varies on the effect of Mark-Houwink’s K and α value. The relationships between the initial Mv and Mw with irradiated Mv and Mw are depicted in Figure 4; Figure 5, respectively.

The initial Mw of the samples was dramatically decreased by the irradiation on all alginate samples. The highest reduction was observed in sample 12 (84% reduction) and the lowest reduction was observed in sample 13 (33% reduction). Samples 2, 4, 5, 7, 8, and 10 yielded approximately 50% reduction due to the effects of irradiation. On the other hand, samples 6 and 9 showed an approximately 60% reduction. In short, all the samples are sensitive towards irradiation (percentage reduction of Mw), but this was also dependant on the dose of radiation. The graphical correlations of molecular and % reduction between the initial and irradiated samples are presented in Figure 6.

According to the calculated DP presented in Table 1, some alginate samples were dramatically affected by the radiation. The results revealed that some samples were sensitive towards a radiation dose of 15 kGy, while several samples degraded comparatively slowly depending on the block of G- and M-units of the alginate samples.

The hydrogel content is observed as acted as a critical nature with the CaCl₂ salt (Figure 7). Among the 26 samples, both in control and irradiated samples showed decreases along with increases, and some are the same in nature in terms of hydrogel content for the binding behavior of alginate to CaCl₂. Based on the established isothermal process, viscosity, and Ca²⁺ selective electrode measurements (Fang et al., 2007), the binding of Ca²⁺ to alginate involved three distinct steps upon increasing the concentration of CaCl₂. The findings were delineated into three distinct and successive steps in the binding of calcium to alginate with increasing concentrations of Ca²⁺: 1) interaction of Ca²⁺ with a single guluronate unit forming monocomplexes; 2) propagation and creation of egg-box dimmers via the pairing of these monocomplexes; and 3) lateral association of the egg-box dimmers, generating multimers. The final step created different association modes based on the varied Mw of the alginate samples.

The interchain association along with the boundaries between these steps were reasonably critical, which closely correlated with the Ca/guluronate stoichiometry outcome for the egg-box dimmers and multimers with 2/1 helical association of the chains. The configuration of the egg-box dimmers and their consequent association thermodynamically corresponded that was fitted by a model of self-regulating binding sites. The relationship of percentage gel content of the initial and irradiated samples is illustrated in Figure 8. The percentage gel contents of the samples in case of gel strength are showed critical behavior in nature. The gel content of samples 2, 3, 5, 6, 9, and 10 decreased after irradiation, while samples 1, 4, 7, 12, and 13 increased, and samples 8 and 11 remained unchanged (initial and irradiated).

On the other hand, the gel strength increased for some samples because more G residues were released in irradiated samples. However, at high doses of irradiation, the alginate samples degraded into smaller units with the existing mechanism (Mollah et al., 2009). Another proposed phenomenon explained that some samples became more sensitive to M residues after irradiation. The M content comparatively decreased, whereas increased G residues quickly reacted with Ca²⁺ to produce egg-box dimmer and increased the percentage gel strength (Figure 7) (Fang et al., 2007). The results also revealed that the isotherm technique which involves three steps of binding phenomena occurred depending on the (Ca/G = R) stoichiometry (step I; R = 0.25, step II; R = 0.55, step III; R = 0.75). Based on the stoichiometry (Figure 8), the samples were laterally associated with egg-box multimers composed of 2/1 helical chains. The egg-box dimmers are formed to laterally aggregate with the inter-cluster association which is related to a reduction in molecular size.

Moreover, the G-break dependent on gel strength was calculated (G-value = gel strength/0.25). The results indicated that the peak in sample 7 (control) was similar to sample 6, while the lowest was identified in sample 13 (control). The G-break was proportional to the gel strength, in accordance with the literature. Hence, it can be inferred that the gel strength depends on the G-value and that it increases in irradiated samples.