Hybrid carrageenans versus kappa–Iota-carrageenan blends: a comparative study of hydrogel elastic properties

Academic Editor: Wei Cui Received: 31 January 2025 Revised: 18 February 2025 Accepted: 20 February 2025 Published: 22 February 2025 Citation: Monteiro, M.A.F.; Faria, B.; Moraes, I.C.F.; Hilliou, L. Hybrid Carrageenans Versus Kappa–IotaCarrageenan Blends: A Comparative Study of Hydrogel Elastic Properties. Gels 2025,11, 157. https://doi.org/ 10.3390/gels11030157 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Hybrid Carrageenans Versus Kappa–Iota-Carrageenan Blends: A Comparative Study of Hydrogel Elastic Properties Maria Alice Freitas Monteiro 1, Bruno Faria 1, Izabel Cristina Freitas Moraes 2and Loic Hilliou 1,* 1Institute for Polymers and Composites, University of Minho, 5800-048 Guimarães, Portugal; [email protected] (M.A.F.M.); [email protected] (B.F.) 2Department of Food Engineering, Faculty of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Postgraduate Programme in Materials Science and Engineering, Pirassununga 13635-900, SP, Brazil; [email protected] *Correspondence: [email protected] Abstract: A comparison between the gel properties of blends of kappaand iota-carrageenans (K+Is) and hybrid carrageenans (KIs) with equivalent chemical compositions is here presented. The objective is to assess under which conditions hybrid carrageenans are valuable alternative to blends of kappaand iota-carrageenans for gelling applications and to contribute to the identification of phase-separated structures or co-aggregated helices. Phase states constructed in sodium chloride and in potassium chloride confirm that KIs build gels under a much narrower range of ionic strength and polysaccharide concentration. Hybrid carrageenans displayed salt specificity, forming gels in KCl but not in NaCl, highlighting their limited gelling potential in Na + environments. A two-step gelation mechanism was found in both systems at lower ionic strengths and when iota carrageenan is the major component. The shear elastic moduli of KI gels are overall smaller than those of blends, but the opposite is observed at lower ionic strengths in KCl and in systems richer in iotacarrageenans. The nonlinear elastic properties of gels do not relate to the use of blends or hybrid carrageenans for their formulation. Instead, larger contents in iota-carrageenans lead to gels able to sustain larger strains before yielding to a fluid state. However, these gels are more prone to strain softening, whereas strain hardening is measured in gels containing more kappa-carrageenan, irrespective of their blend or hybrid structure. Keywords: carrageenan; hydrogel; molecular mass; shear storage modulus 1. Introduction Carrageenans are a family of sulfated polysaccharides, extracted from red seaweeds, which have long been integral to the food industry due to their unique gelling, thickening, and stabilizing properties [ 1 ]. They play a crucial role in the formulation of a wide range of products, including dairy goods, meat products, and plant-based alternatives [ 1 , 2 ]. Carrageenan is currently the leading seaweed-derived food hydrocolloid and accounts for the sixth-largest share of the global hydrocolloid market (in terms of value), after guar gum, gelatin, xanthan gum, cellulose gum, and arabic gum [ 3 ]. The global carrageenan market was worth USD 850 million in 2022 and is expected to expand at an average yearly growth rate of 6.2%, reaching USD 1.55 billion by 2032 [4]. The focus of most commercial applications has been on the use of kappa-carrageenans and iota-carrageenans, either in pure forms or as blends, to achieve desired textural properties [5–7] . Extensive research has been conducted on these carrageenans, particularly in terms of their chemical structure, gelling mechanisms, and interactions with other Gels 2025,11, 157 https://doi.org/10.3390/gels11030157 Gels 2025,11, 157 2 of 18 food ingredients [ 8 ]. However, the increasing demand for kappaand iota-carrageenans in both food and non-food industries [ 9 ] has recently exerted significant pressure on carrageenan production and seaweed farming [ 1 ], making it urgent to look for underexploited and sustainable resources. In this context, kappa/iota-hybrid carrageenans (KIs) [ 10 ], which consist of a random block copolymer of sequences of kappa-carrageenan ( κ ) and iota-carrageenan ( ι ) diads (disaccharides of D-galactose and 3,6-anhydrogalactose units linked by specific glycosidic bonds, see Figure 1), have emerged as promising alternatives to traditional commercial blends. Industrially known as weak kappa or Kappa-2 [ 11 , 12 ], these copolymers form thermo-reversible hydrogels with intermediate elasticity in comparison to those of kappaand iota-carrageenan, offering unique structural and performance advantages in certain applications [ 11 , 12 ]. Moreover, they are predominantly sourced from wild seaweeds in cold waters, unlike kappaand iota-carrageenans, which are mainly obtained from the intense farming of warm-water species, with associated production issues [ 13 ]. The ratio between kappa-carrageenan and iota-carrageenan diads in a KI is set by biology, that is, the family and genus of seaweeds used to extract the KI. So, instead of mixing at different ratios to tune the properties of the blend, the properties of a KI are tuned by choosing the correct seaweed and isolating the corresponding copolymer [ 10 , 14 ]. This makes hybrid carrageenans not only an attractive option for alternative sourcing but also a potentially more cost-effective solution by eliminating the need for blending different types of carrageenans. Gels 2025, 11, x FOR PEER REVIEW 2 of 19 properties [5–7]. Extensive research has been conducted on these carrageenans, particularly in terms of their chemical structure, gelling mechanisms, and interactions with other food ingredients [8]. However, the increasing demand for kappaand iota-carrageenans in both food and non-food industries [9] has recently exerted significant pressure on carrageenan production and seaweed farming [1], making it urgent to look for underexploited and sustainable resources. In this context, kappa/iota-hybrid carrageenans (KIs) [10], which consist of a random block copolymer of sequences of kappa-carrageenan (κ) and iota-carrageenan (ι) diads (disaccharides of D-galactose and 3,6-anhydrogalactose units linked by specific glycosidic bonds, see Figure 1), have emerged as promising alternatives to traditional commercial blends. Industrially known as weak kappa or Kappa-2 [11,12], these copolymers form thermo-reversible hydrogels with intermediate elasticity in comparison to those of kappaand iota-carrageenan, offering unique structural and performance advantages in certain applications [11,12]. Moreover, they are predominantly sourced from wild seaweeds in cold waters, unlike kappaand iota-carrageenans, which are mainly obtained from the intense farming of warm-water species, with associated production issues [13]. The ratio between kappa-carrageenan and iota-carrageenan diads in a KI is set by biology, that is, the family and genus of seaweeds used to extract the KI. So, instead of mixing at different ratios to tune the properties of the blend, the properties of a KI are tuned by choosing the correct seaweed and isolating the corresponding copolymer [10,14]. This makes hybrid carrageenans not only an attractive option for alternative sourcing but also a potentially more cost-effective solution by eliminating the need for blending different types of carrageenans. Figure 1. Chemical structures of the kappa-carrageenan diad (κ) and the iota-carrageenan diad (ι) arranged in sequences in the KI block copolymers or making up the whole polysaccharide chain in the corresponding homopolymers. Despite their promise, these copolymers present several ongoing research challenges. Their chemical structure is influenced by various factors, such as the species of algae, the time and place of harvest, and the parameters and methods used during extraction [14]. These factors do impact KI functionality, as much as they control the gelling properties of commercial kappaand iota-carrageenans [15]. While these variables affect the mechanical and structural characteristics of the resulting hydrogels, the relationship between the gelation mechanism, the gel structure, and the chemical structure of hybrid carrageenans remains complex and poorly understood [10]. Additionally, while the gelling properties Figure 1. Chemical structures of the kappa-carrageenan diad ( κ ) and the iota-carrageenan diad ( ι ) arranged in sequences in the KI block copolymers or making up the whole polysaccharide chain in the corresponding homopolymers. Despite their promise, these copolymers present several ongoing research challenges. Their chemical structure is influenced by various factors, such as the species of algae, the time and place of harvest, and the parameters and methods used during extraction [ 14 ]. These factors do impact KI functionality, as much as they control the gelling properties of commercial kappaand iota-carrageenans [ 15 ]. While these variables affect the mechanical and structural characteristics of the resulting hydrogels, the relationship between the gelation mechanism, the gel structure, and the chemical structure of hybrid carrageenans remains complex and poorly understood [ 10 ]. Additionally, while the gelling properties of commercial kappaand iotacarrageenans are relatively well-documented, their gel structure–elastic properties relationships are far from being fully clarified [16]. Gels 2025,11, 157 3 of 18 Naturally, this is also the case for blends of kappaand iota-carrageenans (here labeled as “K+I”) for which controversial models are proposed (see, e.g., [ 17 ] and references therein). By comparing the experimental results for K+I gel elasticity with blending rules, phase-separated networks [ 18 ] signaled by a two-step gelation [ 19 – 21 ], interpenetrated networks [ 22 ], and co-aggregated networks where helices of kappaand iota-carrageenans are at least partially self-assembling [ 23 ] or form an interactive and associative network [ 24 ] have been proposed. The block copolymer nature of hybrid carrageenans impedes the phase separation of kappaand iota-carrageenan blocks in the helical or coil conformation. Therefore, a systematic comparison of the solution and gel phases of KI and K+I appears as an elegant route to the elucidation of the blend microstructure in the gel phase. Interestingly, this route has not been much explored in the literature, though such a comparison turned out to be a strong argument in favor of the copolymer nature of KI by taking advantage of the ion specificity of kappa-carrageenan in the presence of potassium cations [ 25 ]. Indeed, kappa-carrageenan can be separated from iota-carrageenan in K+I systems prepared in potassium chloride, whereas KIs do not show any phase separation [ 25 ]. KI and K+I comparative phase states in both potassium chloride and sodium chloride were produced for a range of compositions varying between 47 mol.% and 78 mol.% of κ [ 26 ]. These studies were rather application-oriented as the main objective was to screen for regimes of polysaccharide concentration and ionic strength where KIs could be valuable alternatives to K+I gels. Here, the range of blends and copolymer compositions is extended beyond the balanced 50% usually focused on in the literature [ 18 – 25 ], to nearly pure kappaand iotacarrageenans. Following a recent study where a set of hybrid carrageenans were extracted from commercial seaweeds [ 27 ], these copolymers are here alkali-treated to produce KI with virtually no diads of the mu-carrageenan ( µ ) or nu-carrageenan ( ν ) types. These diads are more sulphated than κ and ν diads and are known to limit or impede gel formation [ 10 ]. The large amplitude oscillatory shear (LAOS) behavior of produced KI and K+I gels is reported as it has received less attention in the carrageenan literature [ 10 , 16 , 28 ]. Although LAOS is less frequently used nowadays due to the lack of a unified interpretation of the results [ 29 ], it has given interesting structural characterization when the results are fitted with filament network theories or colloidal gel theory for strain-hardening gels (see, e.g., [16,30–32]). LAOS characteristics are expected to complement the structural information inferred from the gels’ mechanical spectra measured for various polysaccharide and salt concentrations. Overall, the approach set here is expected to contribute to the identification of different gel structures in KI and K+I but also to define the conditions under which hybrid carrageenans can be most effectively used to replace iotaand kappa-carrageenan blends, in particular during industrial processing where large deformations are at play. 2. Results and Discussion 2.1. Characterization of Produced Carrageenans The chemical structures of all extracted and alkali-treated carrageenans are displayed in Table 1. The proton NMR spectra used for the quantitative estimation of the carrageenan diads can be found in Figures S1 and S2 in the Supplementary Materials. All extracted polysaccharides are copolymers, whereas the commercial kappa-carrageenan is a homopolymer since only κ could be detected in the respective NMR spectrum. All samples have no or negligible amounts of µ and ν (given the limit of resolution of proton NMR), with the exception of samples F and B ′ . While the comparison between samples E and F allows for assessing the impact of few ν diads on the phase state, sample D ′ was extracted from another seaweed to replace sample B ′ , since D ′ has the same ι content but with virtually no ν . A certain amount of Floridean starch (circa 10 mol.% of total polysaccharides) was Gels 2025,11, 157 4 of 18 found in samples F, E′, and F′. Therefore, a new KI, sample H′, was produced and treated with NaOH to compare with sample E′and thus assess the impact of Floridean starch. Table 1. Chemical compositions (carrageenan diads from the kappa-carrageenan family in mol.%), molecular mass Mw (in 10 5 g/mol), and polydispersity index PDI of the hybrid carrageenans extracted from commercial seaweeds and alkali modified with KOH or NaOH. The chemical compositions of the commercial kappa-carrageenan (KAPPA) and the commercial iota-carrageenan (IOTA) are also given. Sample KOH Sample NaOH ν µ ι κ Mw PDI ν µ ι κ Mw PDI B 0 0 96 ±5 4 ±2 5.4 ±0.1 4.1 B′1±1 4 ±4 90 ±2 5 ±1 6.1 ±1.5 4.7 C 3 ± 1 0 91 ±1 6 ±2 5.8 ±0.1 1.6 C′0±5 0 95 ±2 5 ±4 8.9 ±1.2 6.2 E 0 0 44 ±6 56 ±4 3.1 ±0.1 2.1 E′1±5 0 42 ±6 57 ±3 4.4 ±0.1 2.8 F 5 ± 5 0 47 ±1 48 ±1 2.5 ±0.1 4.6 F′1±5 0 45 ±5 54 ±3 2.9 ±0.1 2.4 I 0 0 10 ±5 90 ±4 9.6 ±0.3 3.4 I′0 0 10 ±5 90 ±5 7.9 ±0.3 4.5 J 0 0 13 ±3 87 ±5 8.4 ±0.1 2.5 J′0 0 12 ±6 88 ±4 10.6 ±0.1 2.6 K 0 0 5 ±5 95 ±5 5.8 ±0.1 2.9 K′0 0 7 ±5 93 ±4 9.9 ±0.1 2.7 M 0 0 31 ±2 69 ±1 6.5 ±0.1 2.8 M′0 4 ±5 29 ±1 67 ±3 5.3 ±0.1 2.9 KAPPA 0 0 0 100 ±1 11.3 ±0.5 2.7 D′2±2 0 88 ±1 10 ±6 14.7 ±0.2 3.9 IOTA 0 0 92 ±1 8 ±5 9.0 ±0.2 4.7 H′0±5 1 ±5 37 ±6 62 ±1 3.1 ±0.1 2.6 The molecular mass distributions of all carrageenans are shown in Table 1. Interestingly, the type of alkali modification has a strong effect on both the weight-averaged molecular mass Mw and the width of the distribution (PDI) for samples C and C ′ and samples K and K ′ . For these two pairs of samples, the modification by NaOH (samples C ′ and K ′ ) gives hybrid carrageenans with a larger Mw and very different PDI when compared with the polysaccharides treated with KOH (samples C and K). Strong alkali treatments are known to reduce the Mw of carrageenans [ 33 ]. Also, the endogenous salts brought by the dried seaweeds [ 34 ] may add to the alkali modification of the molecular mass distribution. We thus suspect that the seaweeds used to produce samples C and K are rich in potassium salt, thereby increasing the ionic strength of KOH and promoting a larger chain scission than with NaOH. In contrast to this, the remaining hybrid carrageenans show either similar molecular mass distributions (see samples B-B ′ , E-E ′ , and F-F ′ ) or slightly different Mw (see samples I-I ′ , J-J ′ , and K-K ′ ). Mw is known to be a critical parameter for gel formation in carrageenans. In particular, it has been recently shown that the critical mass Mc below which no gel can be formed in KCl depends on the chemical structure of the carrageenans [ 35 ]. Longer iota-carrageenan chains are needed to obtain a gel in 0.1 M KCl at a polysaccharide concentration of 1 wt.% when compared with KI chains. Note here that all Mw values reported in Figure 1are above the Mc found for various hybrid carrageenans and non-commercial kappaand iota-carrageenans [35]. 2.2. Phase States in KCl and NaCl The gel or solution state of all hybrid carrageenans and blends of kappaand iotacarrageenan prepared under various salt conditions and polysaccharide concentrations are presented in Figures 2and 3for KCl and NaCl, respectively. Elemental analysis showed that, due to the alkali treatment, all produced carrageenans brought Na + and K + in amounts varying between 11 and 40 wt.%. Thus, at the largest carrageenan concentrations tested in Figures 2and 3(2 wt.%), the actual ionic strengths are modified by 0.02 M for K + and 0.03 M for Na + , at most. Representative images of the samples’ phases sorted into solutions, suspensions (stable or settled), and gels (with or without water syneresis) are shown in Figure S3 in the Supplementary Materials. Overall, the comparison of the phases in the right and left columns in Figures 2and 3confirmed notable differences between KI and blends [ 12 , 26 ]. Note here that the phase states found for pure iota-carrageenan resemble Gels 2025,11, 157 5 of 18 the phases reported here for the blend with composition 10K+90I, with the exception of low salt conditions (0.01 M), where Michel et al. [ 36 ] reported the absence of gels. As for a comparison of pure kappa-carrageenan with the 90K+10I blend, the same authors found similar phases, except at 0.01 M KCl where pure kappa-carrageenan did not form gels at 0.5 and 1 wt.%, and also in NaCl where pure kappa-carrageenan only formed gels at 2 wt.% for ionic strengths larger than 0.1 M [36]. Gels 2025, 11, x FOR PEER REVIEW 5 of 19 for K + and 0.03 M for Na + , at most. Representative images of the samples’ phases sorted into solutions, suspensions (stable or settled), and gels (with or without water syneresis) are shown in Figure S3 in the Supplementary Materials. Overall, the comparison of the phases in the right and left columns in Figures 2 and 3 confirmed notable differences between KI and blends [12,26]. Note here that the phase states found for pure iota-carrageenan resemble the phases reported here for the blend with composition 10K+90I, with the exception of low salt conditions (0.01 M), where Michel et al. [36] reported the absence of gels. As for a comparison of pure kappa-carrageenan with the 90K+10I blend, the same authors found similar phases, except at 0.01 M KCl where pure kappa-carrageenan did not form gels at 0.5 and 1 wt.%, and also in NaCl where pure kappa-carrageenan only formed gels at 2 wt.% for ionic strengths larger than 0.1 M [36]. Figure 2. Phases formed in KCl by hybrid carrageenans (KI, left columns) and by carrageenan blends (K+I, right columns) with equivalent chemical composition in κ and ι, as a function of 2 factors: polysaccharide concentration (KI or K+I, in wt.%) and ionic strength of K + (in mol/L). Gels (labeled in green in Figures 2 and 3) in the presence of K + occurred more readily in blends than in KI. For ι-rich samples (B and C), gelation was more dependent on the polysaccharide concentration, occurring only at copolymer concentrations ≥ 1 wt.%, indicating a threshold for forming a stable network. Below this concentration, KI samples formed stable suspensions of aggregates, suggesting that the latter are insufficient in number and size to form a fully connected network. Given the high charge density of ι, this Figure 2. Phases formed in KCl by hybrid carrageenans (KI, left columns) and by carrageenan blends (K+I, right columns) with equivalent chemical composition in κ and ι , as a function of 2 factors: polysaccharide concentration (KI or K+I, in wt.%) and ionic strength of K+(in mol/L). Gels (labeled in green in Figures 2and 3) in the presence of K + occurred more readily in blends than in KI. For ι -rich samples (B and C), gelation was more dependent on the polysaccharide concentration, occurring only at copolymer concentrations ≥ 1 wt.%, indicating a threshold for forming a stable network. Below this concentration, KI samples formed stable suspensions of aggregates, suggesting that the latter are insufficient in number and size to form a fully connected network. Given the high charge density of ι , this behavior is expected under low K + concentrations, where monovalent salts like KCl provide enough ionic shielding but not enough for stable gel network formation [ 8 ]. In contrast, all blends formed gels across similar conditions. Gels 2025,11, 157 6 of 18 Gels 2025, 11, x FOR PEER REVIEW 7 of 19 Figure 3. Phases formed in NaCl by hybrid carrageenans (KI, left columns) and by carrageenan blends (K+I, right columns) with equivalent chemical composition in κ and ι, as a function of 2 factors: polysaccharide concentration (KI or K+I, in wt.%) and ionic strength of Na + (in mol/L). Phase labeling is as in Figure 2. Notably, for samples F′, E′, H′, and M′ with 50–70 mol.% κ, no gels formed under any condition, contrasting starkly with the corresponding blends. The differences with samples E′ and F′ in Figure 3 suggest that the distributions of κ and ι blocks as well as their lengths are chemical variables influencing the overall gelling performance of KI in NaCl. The effect of Floridean starch on the gelling ability of KI in NaCl could not be assessed as samples H′ and E′ did not form gels. Overall, as much as the gelling functionalities of carrageenans are at play, the results displayed in Figures 2 and 3 show that hybrid carrageenans can be a valuable alternative to blends of kappaand iota-carrageenan but only at specific salt and polysaccharide concentrations. Nonetheless, rheological testing is essential for a comparative estimation of gel elasticity and resistance to deformation. Figure 3. Phases formed in NaCl by hybrid carrageenans (KI, left columns) and by carrageenan blends (K+I, right columns) with equivalent chemical composition in κ and ι , as a function of 2 factors : polysaccharide concentration (KI or K+I, in wt.%) and ionic strength of Na + (in mol/L). Phase labeling is as in Figure 2. For κ -rich KI samples, ionic strength played a more significant role, with gel formation particularly challenged at elevated K + concentrations ( ≥ 0.5 mol/L for samples J, and >1 mol/L for the corresponding blend). While potassium ions more effectively induce the helical and aggregated states of kappa-carrageenan, salt-out effects at large ionic strength may lead to precipitation, compromising gel formation. A similar high salt sensitivity was observed in samples E and M at a K + concentration of 1 mol/L, where stable suspensions or solutions are observed, whereas low ionic strengths and low polysaccharide concentrations hindered the gelation. Sample F showed difficulty in forming gels, which only occurred at high polysaccharide concentrations (2 wt% KI) and ionic strengths of 0.5–1 mol/L, in evident contrast to the blend with equivalent composition in kappaand iota-carrageenan diads. The content of precursor units in sample F ( ≈ 5 mol.%, see Table 1) may explain such a discrepancy as the presence of ν and µ diads in the KI chains is known for their loss in gel ability [ 10 , 37 ], which is confirmed by the gelling ability of the precursor-free sample E showing similar Mw. Gels 2025,11, 157 7 of 18 Alternatively, the presence of Floridean starch in F could hinder the gel phase seen in the corresponding blend. The existence of a cut-off content in κ (circa 50 mol.%) needed for the gel formation of hybrid carrageenan under specific KCl and polysaccharide concentrations emerges from the differences between sample F and samples E and M, whose phases in KCl compare well with those of the blends and the phase state found for a hybrid carrageenan containing 53 mol.% κ [ 26 ]. The latter study reported that KI produced gels with no water syneresis, in contrast to gels made from the blends. The data in Figure 2confirm the syneresis in blends, but samples E, F, and M are also prone to water expulsion from the network. This discrepancy suggests that possible differences in the Mw as well as in the distributions of κ and ι sequences along the KI chains, which are inherent to the different algal sources used here, give different gel syneresis. Note also that slight differences in the experimental protocols employed (in particular the thermal history during cooling) or in the assessment of water syneresis (subjected to experimentalists’ criterions) can also lead to such discrepancies. In NaCl, the differences between KI and blends are even more striking, which points toward the Na + specificity of these copolymers when they contain between 50 and 90 mol% κ. The gel formation for blends containing more kappa-carrageenan was mostly conditioned by low polysaccharide concentrations, not showing much dependence on the ionic strength (contrarily to what is seen in KCl). As expected, due to the non-specific ion response of iotacarrageenan [ 8 ], ι -rich KI samples (C ′ and D ′ ) demonstrated consistent gelling behaviors in both Na + and K + . In contrast, κ -rich samples (I ′ , J ′ , and K ′ ) required higher polysaccharide concentrations to form gels in NaCl than in KCl. Notably, for samples F ′ , E ′ , H ′ , and M ′ with 50–70 mol.% κ , no gels formed under any condition, contrasting starkly with the corresponding blends. The differences with samples E ′ and F ′ in Figure 3suggest that the distributions of κ and ι blocks as well as their lengths are chemical variables influencing the overall gelling performance of KI in NaCl. The effect of Floridean starch on the gelling ability of KI in NaCl could not be assessed as samples H ′ and E′did not form gels. Overall, as much as the gelling functionalities of carrageenans are at play, the results displayed in Figures 2and 3show that hybrid carrageenans can be a valuable alternative to blends of kappaand iota-carrageenan but only at specific salt and polysaccharide concentrations. Nonetheless, rheological testing is essential for a comparative estimation of gel elasticity and resistance to deformation. 2.3. Gel Formation During Cooling Samples C, E, K, C ′ , and K ′ (sample E ′ did not form gels) were chosen for rheometry, as these differed notably in κ - and ι -carrageenan contents, enabling the investigation of rheological responses tied to the κ -content. This selection also facilitated the evaluation of how polysaccharide concentration and ionic strength influence rheology since these samples formed gels under a broader range of conditions, facilitating direct comparisons within each sample. Given the NMR-related uncertainty in the κ -carrageenan contents in samples C and C ′ , and because 10 wt.% kappa-carrageenan is expected to impact more than 5 wt.% on the gel rheology, a K+I sample with a composition 10K+90I was chosen for comparison with samples C and C ′ . KI from M. stellatus, which resembles samples M and M ′ , has been extensively studied under similar conditions [ 38 – 40 ]. Thus, the rheological characterization of sample E was preferred here. It gives a unique opportunity to explore less-documented carrageenan types, adding depth to the rheological findings, in particular in a composition of κ and ι close to 50 mol.%, which has been much studied in blends of kappaand iota-carrageenan [ 17 – 24 ]. Similar concentrations and ionic strengths were chosen for all selected KI and K+I whenever possible. However, sample K and the Gels 2025,11, 157 8 of 18 corresponding blend did not gel or formed gels with significant water syneresis, precluding any rheological comparison. Thus, a different concentration or ionic strength was chosen. Figure 4gives illustrative thermorheological curves measured during the cooling of hot KI and K+I solutions in order to assess the sol–gel transitions. All cooling curves are available in Figures S4 and S5 in the Supplementary Materials, for gels formed in KCl and NaCl, respectively. Three types of cooling were observed. A clear sol-to-gel transition characterized by the crossover between the shear storage modulus G ′ and the shear loss modulus G ′′ , occurring at the gel transition temperature Tg, can be inferred from the data such as those plotted in Figure 4a,c. Gels 2025, 11, x FOR PEER REVIEW 8 of 19 2.3. Gel Formation During Cooling Samples C, E, K, C′, and K′ (sample E′ did not form gels) were chosen for rheometry, as these differed notably in κand ι-carrageenan contents, enabling the investigation of rheological responses tied to the κ-content. This selection also facilitated the evaluation of how polysaccharide concentration and ionic strength influence rheology since these samples formed gels under a broader range of conditions, facilitating direct comparisons within each sample. Given the NMR-related uncertainty in the κ-carrageenan contents in samples C and C′, and because 10 wt.% kappa-carrageenan is expected to impact more than 5 wt.% on the gel rheology, a K+I sample with a composition 10K+90I was chosen for comparison with samples C and C′. KI from M. stellatus, which resembles samples M and M′, has been extensively studied under similar conditions [38–40]. Thus, the rheological characterization of sample E was preferred here. It gives a unique opportunity to explore less-documented carrageenan types, adding depth to the rheological findings, in particular in a composition of κ and ι close to 50 mol.%, which has been much studied in blends of kappaand iota-carrageenan [17–24]. Similar concentrations and ionic strengths were chosen for all selected KI and K+I whenever possible. However, sample K and the corresponding blend did not gel or formed gels with significant water syneresis, precluding any rheological comparison. Thus, a different concentration or ionic strength was chosen. Figure 4 gives illustrative thermorheological curves measured during the cooling of hot KI and K+I solutions in order to assess the sol–gel transitions. All cooling curves are available in Figures S4 and S5 in the Supplementary Materials, for gels formed in KCl and NaCl, respectively. Three types of cooling were observed. A clear sol-to-gel transition characterized by the crossover between the shear storage modulus G’ and the shear loss modulus G″, occurring at the gel transition temperature Tg, can be inferred from the data such as those plotted in Figure 4a,c. (a) (b) (c) Figure 4. Temperature dependence of shear storage modulus G’ and loss modulus G″ during the cooling of (a) a blend 10% + 90% K+I at 1 wt.% in 1 M KCl, (b) sample E at 2 wt.% in 0.5 M KCl, and (c) sample C at 2 wt.% in 0.01 M KCl. The arrow in (c) indicates the temperature T2 where a second step starts during the gel formation. A monotonic increase of both moduli with the decreasing temperature, where no crossover is detected and thus no Tg, is displayed in Figure 4b. This type of cooling suggests that gelation occurred at temperatures above 85 °C as G′ is larger than G″ in the whole temperature range tested. Note that for sample C′ at 2 wt.% in 1 M NaCl, G′ is always smaller than G″ during the whole cooling down to 25 °C (see Figure S5C). This indicates that sample C′ remained in the liquid state during cooling. The crossover between G′ and G″ occurred during the time spent at 25 °C for the record of the remaining gel properties (see Figure S9C showing G′ > G″ at large strains). Finally, the cooling illustrated in Figure 4c shows two steps in the thermal evolution of G′ (or G″). A temperature Figure 4. Temperature dependence of shear storage modulus G ′ and loss modulus G ′′ during the cooling of (a) a blend 10% + 90% K+I at 1 wt.% in 1 M KCl, (b) sample E at 2 wt.% in 0.5 M KCl, and (c) sample C at 2 wt.% in 0.01 M KCl. The arrow in (c) indicates the temperature T2 where a second step starts during the gel formation. A monotonic increase of both moduli with the decreasing temperature, where no crossover is detected and thus no Tg, is displayed in Figure 4b. This type of cooling suggests that gelation occurred at temperatures above 85 ◦ C as G ′ is larger than G ′′ in the whole temperature range tested. Note that for sample C ′ at 2 wt.% in 1 M NaCl, G ′ is always smaller than G ′′ during the whole cooling down to 25 ◦ C (see Figure S5C). This indicates that sample C ′ remained in the liquid state during cooling. The crossover between G ′ and G ′′ occurred during the time spent at 25 ◦ C for the record of the remaining gel properties (see Figure S9C showing G ′ > G ′′ at large strains). Finally, the cooling illustrated in Figure 4c shows two steps in the thermal evolution of G ′ (or G ′′ ). A temperature T2 (indicated by an arrow) lower than Tg signals the onset of a second gelling process corresponding to the elastic reinforcement (increase in G ′ ) of the gel network established at Tg. Two-step gelation has been reported in K+I (see for instance [ 19 , 20 , 22 , 24 , 41 ]) and in hybrid carrageenan samples [ 42 ]. Tg and T2 are listed in Table 2for all samples tested in KCl and in Table 3for all samples tested in NaCl. In KCl, both ι -rich hybrid carrageenan samples (sample C) and their corresponding commercial blends displayed two-step gelling mechanisms. Despite their high ι -content ( ≈ 90 mol.%), their gelling behavior deviated significantly from the monotonic increase in G ′ and G ′′ typically observed in pure iota-carrageenan [ 23 ]. Because iota-carrageenan typically gels at a higher Tg than kappa-carrageenan, though the difference in Tg depends on the salt type and concentration [ 8 ], the presence of Tg and T2 suggests that even small κ fractions ( ≈ 10 mol.%) can form microdomains or secondary aggregates that reinforce the iota-carrageenan gel network and contribute to a second gelation step. Heterogeneous microdomains related to phase-separated or interpenetrated kappaand iota-carrageenan assemblies have also been evidenced in K+I using scattering techniques [ 18 , 23 ], NMR [18,43], optical microscopy [23], and particle tracking [44]. Gels 2025,11, 157 9 of 18 Table 2. Thermorheological properties of gels formed in KCl. Gel transition temperatures (Tg and T2), gel elastic moduli G0 measured at equilibrium at 25 ◦ C, and large deformation properties (LAOS) quantified by the strain γF for onset on gel-to-fluid shear-induced transition and qualified by the strain-hardening (HARD) or strain-softening (SOFT) behavior occurring at a strain smaller than γF. Gelling Conditions Samples Tg (◦C) T2 (◦C) G0 (Pa) γF(%) LAOS 2 wt.%–0.01 M KCl C 43.6 ±0.2 31.7 ±0.1 2180 ±17 377 ±30 SOFT 10K + 90I 47.6 ±0.3 34 ±2 1022 ±20 118 ±13 SOFT/HARD 1 wt.%–1 M KCl C >85 60 ±2 48.7 ±0.3 331 ±14 SOFT 10K + 90I 79.4 ±0.8 46 ±3 237 ±2 556 ±34 SOFT/HARD 2 wt.%–1 M KCl C 85.4 ±4.3 74.3 ±0.5 985 ±6 404 ±30 SOFT/HARD 10K + 90I >85 - 11,281 ±238 388 ±30 SOFT 2 wt.%–0.01 M KCl E 66.1±0.2 54 ±1 7831 ±23 37 ±5 SOFT 60K + 40I 47.5 ±0.4 39.5 ±0.5 15,351 ±46 60 ±7 SOFT/HARD 1 wt.%–0.5 M KCl E >85 - 2391 ±14 35 ±4 SOFT 60K + 40I >85 - 157 ±1 104 ±10 SOFT 2 wt.%–0.5 M KCl E >85 - 14,860 ±134 11 ±1 SOFT 60K + 40I >85 - 357 ±2 92 ±4 HARD 0.5 wt.%–0.01 M KCl K 43.5 ±1.5 * - 490 ±4 123 ±6 HARD 90K + 10I 32 ±1 * 27 ±1 535 ±5 136 ±7 HARD 0.5 wt.%–0.5 M KCl K >85 - 257 ±2 100 ±9 SOFT 90K + 10I >85 - 1650 ±61 25 ±2 SOFT 2 wt.%–0.5 M KCl K >85 - 20,476 ±71 18 ±2 SOFT 90K + 10I >85 - 81,921 ±4012 3.7 ±0.6 SOFT *: no crossover between G′and G′′, rather the temperature where a step rise in G′occurs. Table 3. Thermorheological properties of gels formed in NaCl. Gel transition temperatures (Tg and T2), gel elastic moduli G0 measured at equilibrium at 25 ◦ C, and large deformation properties (LAOS) quantified by the strain γF for onset on gel-to-fluid shear-induced transition and qualified by the strain-hardening (HARD) or strain-softening (SOFT) behavior occurring at a strain smaller than γF. Gelling Conditions Samples Tg (◦C) T2 (◦C) G0 (Pa) γF(%) LAOS 2 wt.%–0.01 M NaCl C′30.76 ±0.5 29.3 ±0.1 738 ±3 181 ±5 SOFT 10K + 90I 52.0 ±1.5 30 ±1 1400 ±128 155 ±13 SOFT/HARD 1 wt.%–1 M NaCl C′83.9 ±3.4 - 100 ±60 391 ±47 SOFT 10K + 90I 55.6 ±0.3 - 800 ±400 25 ±3 SOFT 2 wt.%–1 M NaCl C′<25 - 80 ±20 355 ±30 SOFT 10K + 90I >85 62 ±3 410 ±50 315 ±38 SOFT/HARD 2 wt.%–0.01 M NaCl K′39.4 ±0.4 40.5 ±0.5 18,226 ±111 36 ±5 SOFT 90K + 10I 44.5 ±0.5 * - 3832 ±140 100 ±5 HARD 1 wt.%–0.5 M NaCl K′53 ±1 * - 3384 ±82 41 ±2 HARD 90K + 10I 46.0 ±0.3 * - 6797 ±45 23 ±1 SOFT 2 wt.%–0.5 M NaCl K′50 ±1 * - 3118 ±96 11 ±4 HARD 90K + 10I 54.7 ±0.3 * - 25,535 ±311 49 ±7 HARD *: no crossover between G′and G′′, rather the temperature where a step rise in G′occurs. Previous studies [ 20 , 22 ] have reported the formation of phase-separated and/or interpenetrating networks in blends, highlighting that κ fractions as low as 2.5% can significantly influence their rheological behavior. Additionally, residual κ -content in the commercial iota-carrageenan (see Table 1) likely amplifies its contribution to network formation in the 10K + 90I blend, raising the actual κ -content to ≈ 17.2 mol.%. This higher effective κ fraction may explain the formation of a secondary gelation phase in these ι -rich systems. However, since Tg and T2 also show up in hybrid carrageenans, a two-step gelation cannot be systematically associated with phase separation. At large compositions in kappa-carrageenan in the blend and κ -content in the hybrid carrageenan, gelling temperatures were only recorded at low salt and carrageenan concentrations, and a single thermal process (Tg) was mainly recorded. Either torque limitation at higher temperatures impedes a clear probe of the networking of the smaller ι fraction or the elasticity of the κ network, building up at temperatures closer to the ιnetwork [8], masks the much softer ιone. Gels 2025,11, 157 16 of 18 and each column shows results at different concentrations and ionic strengths. Top row—sample C vs. K+I 10K+90I at (A) 2 wt.% KI or K+I in 0.01 M KCl; (B) 1 wt.% KI or K+I in 1 M KCl; middle row—sample E vs. K+I 60K+40I at (C) 2 wt.% KI or K+I in 0.01 M KCl; (D) 1 wt.% KI or K+I in 0.5 M KCl; and bottom row—sample K vs. K+I 90K+10I at (E) 0.5 wt.% KI or K+I in 0.01 M KCl; (F) 0.5 wt.% KI or K+I in 0.5 M KCl; Figure S7: Mechanical spectra (storage modulus G ′ , full squares; loss modulus G ′′ , open squares) of hybrid carrageenan (KI) samples (in blue) compared to their respective commercial K+I carrageenan blends (in red) at various polymer concentrations and ionic strengths, in NaCl. Each row represents a different hybrid carrageenan and blend pair: (top) sample C ′ vs. K+I 10K+90I, at (A) 2 wt.% in 0.01 M NaCl; (B) 1 wt.% in 1 M NaCl; (C) 2 wt.% in 1 M NaCl; and (bottom) sample K ′ vs. K+I 90K+10I, at (D) 2 wt.% in 0.01 M NaCl; (E) 1 wt.% in 0.5 M NaCl; (F) 2 wt.% in 0.5 M NaCl; Figure S8: Large amplitude oscillatory shear tests (storage modulus G ′ , full squares; loss modulus G ′′ , open squares, as a function of the applied strain) of hybrid carrageenan (KI) samples (in blue) compared to their respective commercial K+I carrageenan blends (in red) at various polymer concentrations and ionic strengths in KCl solutions. Each row represents a different hybrid carrageenan and blend pair, while each column shows results at different concentrations and ionic strengths. Top row—sample C vs. K+I 10K+90I at (A) 2 wt.% KI or K+I in 0.01 M KCl; (B) 1 wt.% KI or K+I in 1 M KCl; (C) 2 wt.% KI or K+I in 1 M KCl; middle row—sample E vs. K+I 60K+40I at (D) 2 wt.% KI or K+I in 0.01 M KCl; (E) 1 wt.% KI or K+I in 0.5 M KCl; (F) 2 wt.% KI or K+I in 0.5 M KCl; and bottom row—sample K vs. K+I 90K+10I at (G) 0.5 wt.% KI or K+I in 0.01 M KCl; (H) 0.5 wt.% KI or K+I in 0.5 M KCl; (I) 2 wt.% KI or K+I in 0.5 M KCl; Figure S9: Large amplitude oscillatory shear tests (storage modulus G ′ , full squares; loss modulus G ′′ , open squares, as a function of the applied strain) of hybrid carrageenan (KI) samples (in blue) compared to their respective commercial K+I carrageenan blends (in red) at various polymer concentrations and ionic strengths in NaCl solutions. Each row represents a different hybrid carrageenan and blend pair, while each column shows results at different concentrations and ionic strengths. Top row—sample C ′ vs. K+I 10K+90I at (A) 2 wt.% KI or K+I in 0.01 M NaCl; (B) 1 wt.% KI or K+I in 1 M NaCl; (C) 2 wt.% KI or K+I in 1 M NaCl; and bottom row—sample K ′ vs. K+I 90K+10I at (D) 2 wt.% KI or K+I in 0.01 M NaCl; (E) 1 wt.% KI or K+I in 0.5 M NaCl; (F) 2 wt.% KI or K+I in 0.5 M NaCl. Author Contributions: Conceptualization, L.H.; methodology, L.H.; investigation, M.A.F.M. and I.C.F.M.; resources, L.H.; data curation, M.A.F.M. and I.C.F.M.; writing—original draft preparation, M.A.F.M., B.F. and L.H.; writing—review and editing, M.A.F.M., B.F., I.C.F.M. and L.H.; visualization, M.A.F.M. and L.H.; supervision, I.C.F.M., B.F. and L.H.; project administration, L.H.; funding acquisition, L.H. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Fundação para a Ciência e Tecnologia (FCT), through the E2B2-PHACAR project, grant number: PTDC/BII-BIO/5626/2020 (http://doi.org/10.54499/PTDC/ BII-BIO/5626/2020). Additional financial support by the FCT under the framework of Strategic Funding grant: UID/CTM/50025/2020 and grant: CEECINST/00156/2018 is also acknowledged. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The raw data supporting the conclusions of this article will be made available by the authors on request. Acknowledgments: The strong support of Cargill to the E2B2-PHACAR project is acknowledged and also CAPES for the grant awarded to I.C.F.M. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Porse, H.; Rudolph, B. The seaweed hydrocolloid industry: 2016 updates, requirements, and outlook. J. Appl. Phycol. 2017,29, 2187–2200. [CrossRef] 2. Mendes, M.; Cotas, J.; Pacheco, D.; Ihle, K.; Hillinger, A.; Cascais, M.; Marques, J.C.; Pereira, L.; Gonçalves, A.M.M. Red Seaweed (Rhodophyta) Phycocolloids: A Road from the Species to the Industry Application. Mar. 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