The seaweed : water ratio influenced the yield, gel strength, sulfate content, and viscosity because the more significant the ratio of seaweed and the lower the ratio of water, the smaller the solubility and heat transfer that occurs. As a result, less carrageenan will dissolve and escape from the cell walls (Suarez Garcia et al., 2019), because carrageenan is included in the polymer category that dissolve readily in water (Diharmi et al., 2019). As the solvent increases, the number of target molecules interacting with the solvent also increases, resulting in a higher release rate (Wang et al., 2016). The highest gel strength value was for seaweed : water ratio of 15:1 w/v of 117.54 g/cm2.The gel strength value decreases at a higher seaweed : water ratio, and this occurs because as the seaweed ratio increases, the extraction temperature required also increases, causing carrageenan degradation resulting in fragments and decreasing gel-forming ability (Kumar & Fotedar, 2009). The findings of this study are also consistent with the statement by Nurmiah et al. (2017) that the influence of seaweed-to-water ratio and extraction time shows an increase in gel strength up to a specific value. However, further increases in seaweed-to-water ratio and extraction time indicate a decrease in gel strength. The sulfate content and viscosity increase as the seaweed : water ratio increases because the high seaweed ratio causes the distance between particles to decrease, so particle capture by milling becomes less efficient (Borhan et al., 2013). Viscosity increases in direct correlation with sulfate content, indicating that higher seaweed ratios lead to elevated viscosity values and the formation of firmer polymers. The phenomenon is attributed to intensified repulsive forces among negatively charged sulfate groups (Syaharuddin, 2019). Meanwhile, gel strength is inversely proportional to sulfate content. High sulfate levels indicate that more sulfate ester groups bind with water so that the three-dimensional structure formed absorbs more water and can reduce the ability to form gels (Setijawati et al., 2018).
Concentration of celite affects yield, the highest yield value is found at 4% concentration of celite, this increase in yield lies in its ability to filter efficiently and with good synchronization, celite has a structure of tiny pores, low polarity, extensive adhesion, and high porosity (Yudhana et al., 2023) and produces good filtration clarity and has high permeability (Keypour et al., 2013). Celite forms a homogeneous layer over the filter, which helps appropriately separate carrageenan from the solution. Small particles will be trapped in the celite pores when the carrageenan solution is passed through the celite layer. The liquid containing carrageenan will pass through the celite, allowing for more efficient filtration, with more carrageenan being successfully separated from the solution. Using a high concentration of celite, the gel strength will be higher until it reaches a certain point, this occurs because the greater the concentration of celite, the more coarse particles can be captured from the carrageenan solution because more filter material is available. Gain formation can be optimized by removing more contaminants and interfering particles, resulting in stronger gels. Sulfate content and viscosity are negatively correlated with gel strength. However, consistency and gel strength increase as sulfate content decreases. The addition of celite can reduce the sulfate content because the greater the concentration of celite, the viscosity of carrageenan will decrease (Fig. 2d). Consequently, Along the sulfate group polymer chain, there is also a decrease in the repulsive force between negative charges (Ferdiansyah et al., 2023). Viscosity will decrease more as celite concentration rises. This occurs because adding celite to the filter matrix increases permeability, allowing the filter layer to remain solid. The level of compaction correlates directly with permeability characteristics and filter retention. The higher the concentration of celite used, the thicker the compaction, so the better it is at capturing small particles or unwanted solids during filtration (Mukherjee, 2019). By capturing coarse particles and reducing viscosity, celite can allow carrageenan molecules to interact better with each other to form a gel. This can increase gel strength because carrageenan molecules have more opportunities to form hydrogen bonds and other interactions that produce a strong and stable gel structure.
Based on the 3D graph of the response surface, The interaction between the seaweed : water ratio and the celite concentration provides a synergistic effect on increasing yield. The increase in yield is caused by the celite ability to absorb small particles so that they are not wasted during the filtration process, causing the yield produced during the extraction process to be higher (Hakim et al., 2011). In addition, the higher the seaweed ratio, the lower the carrageenan solubility because more seaweed is used than the amount of extraction solution (Rusli et al., 2017) and this is proven by the peak yield value at a seaweed : water ratio of 15:1 w/v, followed by a decrease in the higher seaweed : water ratio of 19:1 w/v. Increasing the seaweed ratio results in inefficient energy distribution during grinding due to weak interactions between the beads and seaweed cells (Borhan et al., 2013), as evidenced by the highest seaweed : water ratio of 27:1 w/v. The yield value has decreased to the minimum point. The interaction between a low seaweed : water ratio and a high celite concentration causes an increase in gel strength. In high celite concentrations, filtration can more efficiently remove unwanted particles from the carrageenan solution. Thus, the resulting gel will have higher purity and a more homogeneous structure, contributing to increased gel strength. Several factors can also influence the strength of the gel, one of which is the sulfate content (Mahyati & Azis, 2019). When the sulfate level of carrageenan is low, the consistency of the gel formed is higher (Siregar et al., 2016) because fewer ester groups bind with water, causing the three-dimensional structure formed to absorb less water and increase gel formation ability (Setijawati et al., 2018). A low seaweed : water ratio and a high celite concentration will produce less viscous carrageenan with a lower viscosity value. The repulsive force of negative charges along the sulfate group polymer chain influences the viscosity of carrageenan. Then, Celite has a highly porous surface and has high adsorption properties for small particles in solution. When the solution flows through the dense celite layer, carrageenan sulfate can attach to the celite surface due to the physical interaction between carrageenan sulfate molecules and the celite surface. Then, as the sulfate content decreases at lower seaweed : water ratios, the depolymerized and degraded sulfate ester polymer chains are shorter (Sandria et al., 2017).
The yield value in the ball mill method with the addition of celite has the highest value compared to without celite treatment and the chemical method (Table 1). The high yield in the ball mill method with the addition of celite is due to a large number of small-sized particles or unwanted solids successfully absorbed by the celite during filtration (Hakim et al., 2011), which allows for more efficient filtration and more carrageenan being successfully separated from the solution. Apart from that, the beads in the ball mill chamber also help grind. The surface area of the cells in contact with the solvent is high because the beads flow or move in a certain way as the chamber moves, colliding with each other to increase yield (Loh et al., 2014). The gel strength of the chemical method has a higher value than the ball mill method because, in the extraction process with alkali, The presence of K+ ions can decrease the negative charge on the polymer chains, creating a three-dimensional structure between them. The three-dimensional polymer structure is strengthened by increasing the ionic strength of the carrageenan polymer chain (Heriyanto et al., 2018). The gel strength of carrageenan using the ball mill method with the addition of celite is reasonably high because dirt and cellulose are successfully filtered by celite. After all, celite has low polarity, broad adhesion, and high porosity (Yudhana et al., 2023).
The sulfate content in the ball mill method with the addition of celite or without celite treatment has a higher value than the chemical method (Table 1). Chemical extraction method with KOH has the lowest sulfate content because alkaline solution can catalyze sulfate group from galactose unit, reducing sulfate content (Subaryono & Utomo, 2006). The viscosity value in the ball mill method with the addition of celite has the lowest value compared to that without the addition of celite and the chemical method (Table 1) because the filtration process using celite can capture small particles, dirt and cellulose, which are also filtered so that the resulting viscosity is low. Meanwhile, the chemical extraction process using KOH has a higher viscosity value because the K+ ions in KOH can dissolve the salt content in seaweed and increase the viscosity value (Anwar et al., 2013).
The melting point and gel point of chemically extracted carrageenan have higher values compared to extraction using a ball mill (Table 1) because kappa-carrageenan is sensitive and can form a helical structure in the presence of K+ ions, thereby increasing the melting point and gel point during the formation process gel (Hamid et al., 2021). The ash content of carrageenan extracted using the chemical method has a higher value than the ball mill method with the addition of celite or without celite treatment (Table 1). Romenda et al. (2013) explained that the presence of mineral salts (such as Na and Ca) attached to seaweed influences the high ash content of carrageenan. The water content value of carrageenan using the chemical method is lower than the ball mill method because the alkaline KOH solution can inhibit the binding of water to the carrageenan molecule, thereby affecting the low water content produced (Wulandari et al., 2019). The pH value of carrageenan extract using the chemical method is higher than the ball mill method with or without celite treatment (Table 1) because the chemical extraction process uses a high-concentration alkali solution. In contrast, the ball mill method uses a water solvent. Carrageenan extracted using the ball mill method with the addition of celite or without celite treatment has the highest degree of purity compared to the chemical method (Table 1), allegedly because the grinding process using a ball mill with centrifugal force causes a large amount of carrageenan to be extracted (Loh et al., 2014). Meanwhile, carrageenan chemically extracted using KOH has the lowest degree of purity because carrageenan can experience degradation due to alkaline solutions (Distantina & Fahrurrozi, 2011).
FTIR analysis results (Fig. 4) show a band at wave number 3448.72 cm− 1 in the ball mill method without celite treatment and 3425.58 cm− 1 in the ball mill method with celite treatment, indicating the presence of hydroxyl groups. The 3200 − 3500 cm− 1 range FTIR spectrum shows hydroxyl groups (O−H) (Akindoyo et al., 2017). Furthermore, the absorption wavelength was 2924.09 cm− 1 in the ball mill method without celite treatment and with celite treatment, indicating the presence of alkane compounds and carboxylic acids (Hari et al., 2020). The subsequent absorption wavelength of 2376.30 cm− 1 indicates the presence of a phosphine group (Soltani et al., 2016). Meanwhile, at a wavelength of 1620.21 cm− 1, it shows the presence of amine and alkene groups (Hari et al., 2020). Furthermore, the treatment without celite has an absorption wavelength of 1381.03 cm− 1, while with the celite treatment, it has a wavelength of 1373.32 cm− 1, indicating the cutting characteristics of –CH2 in the carboxymethyl group (Eliza et al., 2015). Intense absorption in the ball mill method with celite treatment occurs at a wavelength of 1234.44 cm− 1, while without celite treatment, it occurs at 1249.81 cm− 1. This wavelength absorption indicates the presence of a sulfate ester group (Rhein-Knudsen et al., 2017). The next absorption band is the glycosidic bond, which has a wavelength of 1049.28 cm− 1 for the ball mill method without celite treatment and 1072.42 cm− 1 with celite treatment. The wave number 925.83 cm− 1 indicates the 3,6-anhydrogalactose group's existence (Al-Nahdi et al., 2019), the absorption peak of the 3,6-anhydrogalactose group is at 925 − 935 cm− 1. Furthermore, both treatments had an absorption wave number of 848.68 cm− 1, which is the absorption of the galactose 4-sulfate bond, where this bond indicates that the carrageenan produced is included in the kappa-carrageenan type (Bhernama, 2019).
Gel strength increases with higher KCl concentration. The ionic strength of the polymer chain can be strengthened by the K+ ion in KCl, causing the intermolecular forces to become higher and the balance between the ions bound in the carrageenan to form a gel (Hakim et al., 2011). the higher the concentration of KCl results in an increase in viscosity. Due to its status as a polyelectrolyte, carrageenan has an increased viscosity. The molecular chain becomes rigid due to the force that repels the sulfate groups, which are the negative charges along the polymer chain. Polymers that are surrounded by water molecules are unable to move due to their hydrophilic nature, resulting in the formation of a thick solution of carrageenan (Wullandari et al., 2021). In addition, forming 3,6-anhydro-galactopyranose due to alkaline conditions caused by K + can increase the viscosity and strength of the gel (Jayasinghe & Pahalawattaarachchi, 2016). The higher the KCl concentration, the melting point and gel point of carrageenan also increase. The capacity of carrageenan to form a network through a coil-helix transition and then aggregate, caused by the presence of ions, increases the gel and melting points (Loret et al., 2009). Kappa-carrageenan is sensitive and can form a helical structure in the presence of K+ ions, thereby increasing the melting point and gel point during the gel formation process. During the heating process, hydrogen bonds are broken, and the double helix structure will form aggregation and then separate. At the endothermic transition temperature during the cooling process, a helical structure will form, and at a particular melting temperature, gel formation will take place. Thermal hysteresis is the name given to this discrepancy between the melting and gel points, where double helix aggregation occurs, leading to gel formation (Hamid et al., 2021).
The strength of the gel with added CaCl2 is lower than without the addition of CaCl2, and this is because Ca2+ ions are added to the carrageenan solution. These ions can disrupt the network formed by replacing the bonds between the carrageenan chains or by replacing the carrageenan groups that have bonded with water. As a result, the gel structure becomes looser, and its strength decreases. Figure 6b The addition of CaCl2 appears to increase the viscosity of carrageenan. However, the effect is not significant because the type of carrageenan found in the seaweed Kappaphycopsis cottonii is kappa-carrageenan (Nurani et al., 2024). Meanwhile, the cation that plays a role in the gelation of kappa-carrageenan is the K+ cation, not Ca2+, because the Ca2+ cation is more effective in causing gel formation in iota carrageenan (Diharmi et al., 2015). When cooled, the intermolecular bridges formed by Ca2+ cause the carrageenan solution to form a quaternary structure. On the contrary, by creating an ionic link with the sulfate group on the D-galactose residue, K+ will cause the combining of kappa-carrageenan molecules. The anhydro oxygen atom of the adjacent galactose residue and the K + will then form a secondary electrostatic bond as a result of this process (Thrimawithana et al., 2010). The increase in melting point and gel point is caused by the ability of Ca2+ cations to facilitate coil helical transitions to form wider and more aggregates, so that they require greater energy to melt (Liu & Li, 2016).