Cross-linking Formation of Taro Starch (Colocasia Esculenta)-Based Hydrogel using Freeze-Thaw Method: Synthesis and Physical Characterization

Materials

Taro flour was obtained from CV Primanaya, Indonesia for taro starch isolation. The distilled water and sodium metabisulfite (Na₂S₂O₅, food grade) were purchased from PT Brataco Chemistry, an Indonesian supplier. The phosphate-buffered saline (PBS) solvent was acquired from the School of Pharmacy ITB (Institut Teknologi Bandung) in Bandung, Indonesia.

Taro Starch Isolation and Hydrogel Synthesis

Starch isolation was carried out with reference to Ahmed et al. 24 and Vithu et al 20, with slight modifications, including an immersion method using distilled water (AQ), sodium metabisulfite (SM), and a centrifugation method (CE). SM and AQ methods were simple with a 2:5 (w/v) blend of taro powder in 1% Na₂S₂O₅ solution and distilled water (10 g/30 mL). After cheesecloth filtering, the liquid was sedimented for 24 hours at room temperature. All suspended impurities were removed using distilled water, and the residue was dried at 50°C for 12 hours to make starch powder. The CE method used a 3500-rpm centrifuge to separate the sediment. The dry powder, after centrifugation, was crushed in a mortar and screened through an 80-mesh screen for fine taro starch powder. The isolation process of taro starch was summarized in Figure 1.

Taro starch hydrogel was started by optimizing the freeze-thaw process parameters, including freezing-thawing time for 16-8 hours (a), 17-7 hours (b), 20-4 hours (c), respectively, with freezing/thawing temperature of samples a and b of -23/4°C, while sample c was -23/37°C. The great freeze-thaw parameters (the highest swelling degree, the lowest weight loss) were then fabricated with variations in taro starch concentration of 7%, 10%, and 13% (w/w) in different isolation methods (AQ, SM, CE). Starch was dissolved in distilled water and stirred at 100°C for 2 hours. After viscous and gelatinization, the precursor solution was frozen and thawed with parameter b, resulting from optimizing before: freezing for 17 hours at -23 °C and thawing for 7 hours at 4 °C. This freeze-thaw process was done for four cycles. The schematic of taro starch hydrogel synthesis is illustrated in Figure 2a.

Characterization of Taro Starch

Taro starch paste was characterized and modeled using Rapid Visco Analyzer (model RVA-4, Newport Scientific, Australia) with Standard Analysis 1 profile. The paste parameters obtained were pasting viscosity (PV), holding viscosity (HV), pasting temperature (PT), final viscosity (FV), breakdown viscosity (BV), and setback viscosity (SV). For mathematical modeling analysis, according to Palabiyik et al., in the four model parts 26: The first part, Eqn (1)’s modified Hill model, explains the first part of the curve’s beginning area to PV.

V is viscosity (mPa.s), t is time (s), R is time to reach 50% of PV (s), and S is starch coefficient, which measures starch swelling. S>1 implies that water entry into starch granules accelerates swelling. If S<1, starch granule swelling is prevented, and if S=1, water infiltration does not cause swelling.

The second component is explained by Eqn (2)’s Arrhenius model. It’s in PV till the temperature peaks before stabilizing.

η,A₀, E_a,R, dan T represent viscosity (mPa.s), Arrhenius model constants, gas constant (8.314 J/mol.K), and temperature (K). The third part is the exponential model in Eqn (3), which explains the steady temperature third component. η represent viscosity (mPa.s), t represent the time during the third part model (s), K and n are model parameters.

The fourth part is similar to the second part model with Eqn (2).

The formability of the hydrogels was evaluated using an optical microscope, SEM, swelling degree, weight loss, color analysis, and TPA. The internal morphology of the hydrogels was analyzed using an optical microscope and SEM by cutting the hydrogels transversely. Magnification of 10x was performed using AmScope MUI1000, United States, and 5000x was performed using SEM (JEOL, JSM-6510LA, USA).

The swelling degree was also investigated to see the behavior of the hydrogels when exposed to liquids. This test was conducted by immersing the hydrogel in a phosphate-buffered saline (PBS) solution for 48 hours. Previously, the hydrogel was dried for three days at 50°C to evaporate the water, then weighed and referred to as W₀. Throughout the testing procedure, the hydrogel was weighed at intervals, precisely at 0, 3, 6, 9, 12, 24, and 48 hours, and labeled as W_t. The ratio between the weight before and after immersion is calculated as the swelling degree shown by Eqn (4).

The soaked hydrogel was then dried again for three days at 50 °C and weighed as W₁ to see the weight loss of the starch hydrogel. Weight loss can be calculated by Eqn (5).

The color analysis of starch hydrogel was analyzed using starch photographs, using the bivariate histogram approach with the PULUZ foldable LED Ring Light Studio and Nikon D3100 camera. The software used for analysis was ImageJ (version 64-bit Java 1.8.0.172, NIH, USA), which provided white-level histograms 27.

The texture of the hydrogel can be analyzed using TPA tests conducted with a texture analyzer (TA. XTExpress, Stable Micro Systems). The hydrogel samples were dehydrated using a dehydrator at 50°C for 12 hours. This test was conducted by pressing the hydrogel using a cylindrical P36 probe with a diameter of 36 mm so that the amount of adhesiveness, hardness, springiness, cohesiveness, gumminess, chewiness, and resilience could be obtained.

Pasting Properties of Taro Starch

Starch gelatinization is one of the interesting characteristics to be discussed. During the gelatinization process, a phase transition occurs that is stimulated by the starch suspension being heated in water, resulting in granule swelling. This reduces crystallinity and releases amylose from the starch granules. The resulting hydrogen bonds between the OH groups of amylose and amylopectin, which change the properties of the paste, as shown by the amylograph curve (Figure 3) 40. Amylograph characteristics are influenced by several factors, including granular morphology, starch content (amylose/amylopectin), and non-starch components such as phosphorus, lipids, and proteins 41.

Figure 3a shows the curve of the change in viscosity of starch against time during temperature increase and decrease. This curve shows the characteristics of taro starch with different isolation methods. Pasting temperature (PT), pasting viscosity (PV), holding viscosity (HV), final viscosity (FV), breakdown viscosity (BV), and setback viscosity (SV) represent the properties of the starch paste 42. The specific values of the starch paste properties are summarized in Table 2. The PT is the temperature at which the viscosity of starch starts to rise, indicating the onset of starch granule gelatinization. Due to increased temperature, water is absorbed into the starch granules in the gelatinization process. The higher moisture content was the higher the PT, which indicates a longer absorption process. The SM method showed the highest moisture content of about 13.6% and the highest PT value. The sample with the AQ isolation method showed the lowest moisture content of about 10.46% 31,36.

PV is influenced by fat and starch content. During gelatinization, the presence of fat leads to the formation of starch-fat complexes around the granules, making it more difficult for water to be absorbed into the granules 41. Furthermore, the PV value is influenced by the starch content as the rise in viscosity during the gelatinization process is a result of the swelling of starch granules 43,44. The AQ method with the lowest lipid and highest starch content has the most significant PV, suggesting a more rapid gelatinization process.

The parameters SV and FV are related to the retrogradation process of starch granules. SV indicates that the starting point of viscosity increases again during the retrogradation process. Meanwhile, FV relates to the final viscosity after the process. Both describe the retrogradation ability of starch, which depends on the starch content (amylose and amylopectin) 26. The AQ method with the highest SV and FV values demonstrated the most uncomplicated retrogradation process. The retrogradation process includes the cross-linking of molecules that contain amylose and amylopectin.

HV is the viscosity of starch granules disrupted during shearing and heating. Starch granules become increasingly susceptible to shear and heat disintegration as they swell, especially in starches with lower amylose content 45,46. Amylose is released from the granule during the gelatinization process. When amylose is small, the susceptibility of the granule to disintegration is smaller, as shown in the sample using the SM method. BV is correlated with swelling capacity, temperature stability, and the ability of starch granules to maintain their structure 47. AQ, with the highest BV value, shows a firmer structure and superior starch granule integrity. In contrast, SM, with the lowest BV value, exhibits the lowest strength and stability. This is directly proportional to the starch content in each sample, according to the reference in Table 2.

The area under the viscosity versus time curve (Figures 3b-c) depicts the state of the starch sample from liquid-paste-gel during the gelatinization and retrogradation processes. It can be helpful to control the starch product by considering the amylose and amylopectin content 48,49. The AQ sample with the highest starch content showed the highest area of 139.58 Pa.s², indicating the stability of the sample in paste form. On the other hand, in the SM sample, the shape of the sample changes quickly to either liquid or solid again.

Simple mathematical modeling was conducted to analyze product development and starch characteristics, such as understanding how quickly the viscosity peak is reached to determine the processing time for product manufacturing. In addition, it can understand the swelling kinetics of starch granules during the gelatinization process and predict various parameters such as starch texture and rheological properties, which are helpful for quality control and product processing design 26,43. The pasting curve is segmented into four sections of mathematical models and quantitatively shown in Table 3. In the initial model, the R-value represents the time at which the viscosity hits 50%. This measurement is useful for examining the resistance of the starch to gelatinization and retrogradation processes 43. The AQ method with the highest starch content exhibited a higher duration for granule disintegration, measuring 309.88 s, followed by CE and SM at 306.92 s and 304.59 s, respectively. The S-value in this model is the starch coefficient, which indicates the swelling rate. Regarding our result, the entry of water into the starch granule increases the swelling rate of the granules as S>1 for all starch samples. The SM method has the highest S of 26.47, followed by CE and SM of 16.64 and 16.42, respectively. These findings are consistent with prior research, which found that starch amylose content is inversely related to S-value. R-value and S-value are related to the gelatinization process, which involves hydrogen bond breaking in the amorphous part and water absorption related to granule swelling in the amorphous region 26,50.

The second model shows granule degradation as the temperature increases. The curve obtained from this study did not fit the model because the granule degradation indicated by the viscosity decrease occurred when the temperature was already stable. This phenomenon aligns with prior studies finding that in corn and wheat samples that have reached a stable temperature state at maximal viscosity, it is not fit using this model 26. In the third model, the K-value relates to the viscosity influenced by amylopectin 51. The high content of amylopectin leads to reduced stability of starch as the swollen granules during stirring and heating 26. The SM method with the highest amylopectin content and the lowest amylose content has a K-value of 15.33 Pa.s, indicating the lowest starch stability. The coefficient of determination (R²) for this model varied between 0.9817 and 0.9954, which implies a high level of accuracy in fitting the curve presented in Figure 3a.

In the fourth model, Ea correlates with the rate of retrogradation. Upon cooling, amylose molecules within the starch granule interact with amylopectin, resulting in the formation of a solid structure 26. The sample with the highest amylose concentration exhibited the highest activation energy (Ea) value and retrogradation rate.

Macro-microscopic Analysis of Taro Starch Hydrogel Formation

The macro-micro properties of hydrogel formation are influenced by the amylose-amylopectin ratio. The ratio of amylose/amylopectin is crucial in determining the textural properties and stability of starch-based hydrogels. The high amylose content in hydrogels forms dense and rigid structures with low water absorption and increased stability, making them ideal for applications requiring firm and durable gels. During freezing, amylose chains released from the granules are compressed by ice crystals, promoting the formation of hydrogen bonds. Upon thawing, the remaining free hydroxyl groups establish additional bonds (Figure 2b). Hydrogen bonds of the linear amylose chains form a cross-linking hydrogel network (Figure 2c). In contrast, hydrogels with higher amylopectin content have a more porous structure, facilitating enhanced water absorption and resulting in softer, less firm gels that tend to disintegrate more easily in water 26,39. These differences allow the amylose-amylopectin ratio to control properties suitable for various applications.

×: no hydrogel formed; o: hydrogel formed with brittle structure; ✓: hydrogel formed with firm structure.

Figures 4 show the freeze-thaw parameter optimization of CE hydrogel using swelling degree and weight loss analysis. A higher degree of swelling and weight loss represents good structural integrity as a delivery platform. The concentrations used were 10% and 13% (w/w). The swelling degree is related to the hydrogel’s ability to swell, which can be used in the loading and controlling release of nutraceuticals 52, and can help reduce hunger by enlarging as it traverses the digestive system 48. Swelling can be tuned through process parameters including polymer composition, cross-linking density, porosity, and external stimuli (pH, ionic strength, temperature). The advantage of swelling-controlled release is that it allows for tuning of the release profile by adjusting the hydrogel design parameters 52. While weight loss reflects the structural stability and degradation profile of the hydrogel. Controlled degradation is essential for transient delivery systems, where excessive weight loss ensures network erosion and can compromise structural integrity and lead to burst release, which is undesirable for precise dosing 9. According to our results, the sample with the parameters of freezing for 17 hours at -23 °C and thawing for 7 hours at 4 °C produced the highest swelling degree and the lowest weight loss, which are the most optimum parameters for hydrogel formation. In contrast, freezing for 20 hours at -23 °C and thawing for 4 hours at 37 °C showed the lowest swelling degree and the highest weight loss. The swelling degree and low durability of the hydrogel structure at 37°C thawing conditions were caused by the extreme temperature during the thawing process. At the same time, the starch chain (amylose/amylopectin) is a long polymer chain that requires a longer cross-linking process to produce a stronger cross-link at a lower thawing temperature (4 °C) 53.

Moghadam et al. (2022) reported that a starch-based hydrogel as a drug delivery system, composed of PEG and loaded with quercetin, showed a swelling degree of 103-140% 54. Another study reported a swelling degree of starch/polyacrylamide/gelatin composite hydrogels of around 500% 55. In this study, the taro starch hydrogel synthesis process is presented without adding other polymer reinforcements, and it is an environmentally friendly method. Taro starch hydrogel successfully achieved a swelling degree of 150-500%. The highest swelling degree is helpful for the controlled release of nutraceuticals, so taro starch hydrogel has the potential as a delivery system.

Table 4 shows the formation of hydrogels macroscopically with different starch isolation methods and different starch concentrations. Hydrogels formed with brittle structures have a swelling degree of less than 12 hours, while hydrogels formed with firm structures can swell beyond 12 hours. The viscosity of the paste has a significant impact on the formation of hydrogels during the gelatinization process. However, in this case, the sample has not thickened sufficiently to become a paste. Amylose, a fundamental factor in the formation of hydrogel structure, significantly impacts the viscosity throughout the gelatinization process 41. In the CE isolation method, formation began at a starch concentration of 7%, while in the AQ and SM isolation methods, formation began at a starch concentration of 13%. The CE isolation method might have a higher amylose content (in reference to Table 2) as a cross-linking agent for the hydrogel structure.

Figure 5 shows the hydrogel formation process to explain Table 4 with a microscopic analysis. The formation of hydrogel, shown in the morphological structure of optical microscope and SEM images, is divided into three phases: granular swelling, potential cross-linking, and cross-linking phases. The granular swelling phase indicates unformed hydrogels (Figure 5a), where starch granules expand due to gelatinization. These phases are observed in samples with SM and AQ isolation methods at 7% and 10% concentrations. As studied in previous research, swelling of granules occurred in native starch that was not prepared with the cross-linking process 24,56. In addition, the potential cross-linking phase indicates that hydrogels formed with a brittle structure have begun to show porosity (Figure 5b). These potential cross-linking consist of amylose at low concentrations, where the O-H bonds are weak to form stable bindings. The potential cross-linking phases were observed in samples with AQ and SM isolation methods at a concentration of 13% and in samples with the CE isolation method at a concentration of 7%. Finally, hydrogels with 10% and 13% starch concentrations demonstrated that the cross-linking structure phase in the CE isolation method forms hydrogels with a firm structure (Figure 5c).

The formation of the hydrogel structure is influenced by amylose as a linear chain and amylopectin as a branched chain. During gelatinization, amylopectin expands into a large gel-like structure, often referred to as a "gel-ball" or "super-globe," while amylose diffuses out of the granule. During the freeze-thaw process, the amylose chains then cross-link through interactions between hydroxyl (O-H) groups, enhancing the gel network structure 39,57,58. Cross-linking between the O-H groups of amylose in the freeze-thaw process occurs with the help of ice crystals that push the polymer chains to bond and reorganize during thawing 53. Typically, the freeze-thaw method is used for OH-rich polymers such as polyvinyl alcohol 10,11,12, 59,60,61. However, by tuning the process parameters, freeze-thaw can be used to synthesize OH-rich natural polymers such as starch. Several previous studies have developed freeze-thawed starch hydrogels by adding synthetic polymers or cross-linkers as reinforcement 39,62,63. These taro starch hydrogels, with entirely natural polymers without synthetic polymer reinforcement, and non-toxic solvents and processes, successfully formed cross-linked hydrogels and porous hydrogel structures. Prior research has indicated that hydrogels with cross-linked and porous structures have the potential to be a delivery platform for nutraceuticals. Porosity determines the structural design of hydrogels, controlling nutraceuticals loading, diffusion pathways, and swelling kinetics. However, high porosity increases loading and accelerates release, compromising mechanical stability. Therefore, a balance between permeability and structural integrity is essential for reliable delivery performance 64.

Figure 6 shows the swelling degree analysis to confirm the quantitative results under the morphological analysis. The swelling degree is a critical characteristic for practical applications as a potential delivery matrix. The three phases of hydrogel formation are indicated by a blue background for the granular swelling phase, a green background for the potential cross-linked phase, and an orange background for the completely cross-linked phase. The granular swelling phases are shown by samples SM (7%, 10%) and AQ (7%, 10%), which can swell in less than 3 hours. The potential cross-linking phases are shown by AQ (13%), SM (13%), and CE (7%), which can swell up to 12 hours, while the cross-linking phases are shown by samples CE (10%, 13%), which can swell up to 24 hours.

Overall, cross-linking formation of hydrogel is influenced by: (1) starch content, especially amylose which is the main cross-linking factor of hydrogel structure, (2) impurities/ash content, which makes it more difficult for OH to bond, (3) protein, which can help cross-linking through electrostatic interaction, hydrogen bonding, and Van der Waals interaction 65,66. Samples prepared with the CE method showed the highest amylose content, allowing potential cross-linking to form at a concentration of 7%. Meanwhile, in samples with AQ and SM methods that show lower amylose levels than CE, potential cross-linking is formed at a starch concentration of 13%. Furthermore, the high protein levels and low ash content affect the process of hydrogel cross-linking in CE samples.

The whiteness of taro starch hydrogels serves as an indicator of the purity of starch. It can validate the test results on the ability of hydrogel formation. Color analysis is crucial in the food industry as it helps determine the freshness, ripeness, diversity, and appeal of food products, ultimately leading to higher consumer acceptability 67,68. Starch whiteness corresponds to amylose content 39 and is a sensory color characteristic that the industry desires 69. Figure 7 shows the highest whiteness level in the CE sample at 163.34, followed by AQ and SM at 143.45 and 132.24, respectively. This is directly proportional to the amylose content shown in Table 1. The amylose chain is a linear structure that prevents the formation of colors or other substances that cause a decrease in whiteness 69.

Texture Properties of Taro Starch Hydrogel

The textural properties of taro starch hydrogels were characterized using dried hydrogels (Table 5). Hardness is the mechanical resistance of a material, while fracturability is the ability of a material to crack or crumble. Both parameters increased in hydrogels with concentrations of 7% and 10% while decreasing at 13%. The decrease in hardness and fracturability was caused by excessive glucose unit length in taro starch amylose and inhibited the O-H cross-linking process 39. The observation indicates that the weight loss was lower when the concentration of taro starch was 13% compared to 10% (Figure 4. Likewise, chewiness and springiness, which are related to the elasticity of the hydrogel, decreased at 13% concentration. The decrease in these parameters was confirmed by the results of the swelling degree test in Figures 4a-b, which show an up-and-down graph (unstable) during the 24-hour test. The swelling degree is the ability of cross-links to stretch (elasticity) and is influenced by water trapped in the pores of the hydrogel structure 9. Likewise, chewiness and springiness, which are related to the elasticity of the hydrogel, decreased at 13% concentration. The decrease in these parameters was confirmed by the results of the swelling degree test in Figures [FIGREF:4]a-b, which show an up-and-down graph (unstable) during the 24-hour test. The swelling degree is the ability of cross-links to stretch (elasticity) and is influenced by water trapped in the pores of the hydrogel structure 39,70. However, the cohesiveness, gumminess, and resilience reduced as the concentration of taro starch. The decrease in these parameters is attributed to the elevated concentration of taro starch, which inhibits granular swelling and consequently prevents cross-link formation in the hydrogel network structure. The gel network structure is weaker after the concentration is increased. The findings align with prior studies 17, indicating that elevating the corn starch concentration reduced cohesiveness, gumminess, and resilience.

The texture properties of the taro starch hydrogel obtained in this study showed distinctive characteristics compared to other natural and starch-based polymer hydrogels reported in several previous studies. Wu et al. (2017) reported that the hardness of quinoa and corn starch hydrogels was 201–900 g and around 721 g, respectively 71. Additionally, the hardness of pea starch hydrogels with alcohol solvents is 928–1696 g 72. The hardness of taro starch hydrogels reached 855–1752 g, indicating that hydrogels can achieve greater cross-linking stiffness. Regarding chewiness 15, corn starch hydrogels with xanthan gum or sodium alginate reported values between 30–164 g, while taro starch hydrogels were in the 58–156 g range. Taro starch hydrogels synthesized using the freeze-thaw method without adding other polymer reinforcements and toxic solvents showed comparable mastication resistance to composite hydrogels. Thus, taro starch has the potential as a single biopolymer for hydrogel formation, offering a simpler and more environmentally friendly alternative with competitive texture performance and good structural integrity.