Hybrid carrageenans as alternatives to commercial blends: rheological properties and performance in a food application

University of Minho School of Engineering Maria Alice Freitas Monteiro Hybrid Carrageenans as Alternatives to Commercial Blends: Rheological Properties and Performance in a Food Application January 2025 Hybrid Carrageenans as Alternatives to Commercial Blends: Rheological Properties and Performance in a Food Application Maria Alice Freitas Monteiro UMinho | 2025 University of Minho School of Engineering Maria Alice Freitas Monteiro Hybrid Carrageenans as Alternatives to Commercial Blends: Rheological Properties and Performance in a Food application January 2025 Master’s Dissertation Master’s in Chemical and Biological Engineering Dissertation supervised by Prof. Dr. Bruno Faria and Prof. Dr. Loïc Hilliou ii COPYRIGHT AND TERMS OF USE FOR THIRD PARTY WORK This dissertation reports on academic work that can be used by third parties as long as the internationally accepted standards and good practices are respected concerning copyright and related rights. This work can thereafter be used under the terms established in the license below. Readers needing authorization conditions not provided for in the indicated licensing should contact the author through the RepositóriUM of the University of Minho. License granted to the users of this work: CC BY https://creativecommons.org/licenses/by/4.0/ iii ACKNOWLEDGMENTS I would like to dedicate this work to all those who have been a part of this journey and have somehow contributed to the successful completion of this important milestone. First and foremost, I am deeply grateful to my parents for their unwavering support and encouragement throughout this journey. Through their dedication, hard work, and perseverance, they have shown me what it truly means to stay committed and resilient in the face of challenges. Their example of kindness, integrity, and selflessness have been my guiding light, and I am forever grateful for the values they have instilled in me. To my sister, Margarida, I would like to express my heartfelt gratitude, for her belief in me has been a source of strength during challenging times. I truly admire how she fiercely defends her beliefs yet embraces change and seeks out what makes her happiest, always looking out for others along the way. Thanks to her, I am not afraid to aim for the stars, as I have seen her conquer the universe. To the rest of my family, João, Conceição, Joana, Luís, and to my favorite little humans in the whole world, Mafalda and Filipe, thank you for your unconditional support and for inspiring me to always be a better person. To my number one fan – Alex, thank you for always seeing the best in me and for making sure I saw it too. Your encouragement, reassurance and loving words kept me going in times I felt like giving up. It has been my greatest pleasure sharing this life with you. To my beautiful friends, Márcia, Filipa, Inês, Margarida and Gabriela, I leave my profound gratitude, for sharing this adventure with you has been the most rewarding and fun part of my journey. A special thanks to Gabriela for all the motivation and partnership during this 6-month internship – life at the lab wouldn’t have been the same without your companion, humor and so much needed help. To my longlasting friend Guida, who isn’t yet a teacher, but has already taught me so much about perseverance, courage, kindness and compassion – you are truly an inspiration. I wish them all the success and happiness in the world. To Professors Bruno Faria and Loïc Hilliou, I am grateful for all your support, feedback and shared knowledge throughout this project, and for trusting me with this academic enriching opportunity. Also, a special thanks to Professor Germán Valencia for the valuable and unmatchable contribution to this work. To Professor Izabel Moraes, whom along the way I got the pleasure to consider my friend Bel, my most iv heartfelt gratitude. It was a great honor to work with such a gentle and considerate person, and to learn so much from you. I will always carry your kind words with me. Last but not least, I would like to express my sincere gratitude to all the DEP and IPC staff for welcoming me so well, especially to Rui, whom since day one went above and beyond to help me get through this challenge. Thank you for all the tips, motivating and friendly words and for making the dayto-day in the office so lively. This work was supported by the Fundação para a Ciência e Tecnologia (FCT), through the E2B2PHACAR project (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 are also acknowledged. We are grateful to Cargill (Cargill France SAS) for supporting this study by suggesting research topics and providing the E2B2-PHACAR project with commercial seaweeds. v STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. University of Minho, Braga, January 2025 Maria Alice Freitas Monteiro vi RESUMO Carrageninas Híbridas como Alternativas a Misturas Comerciais: Propriedades Reológicas e Desempenho numa Aplicação Alimentar O principal objetivo deste projeto foi comparar as propriedades reológicas e os mecanismos de gelificação de carrageninas híbridas (KI) e misturas comerciais (K+I), com composição equivalente de κ, sob diferentes concentrações de sal e de polissacarídeo. A caracterização das carrageninas extraídas incluiu ¹H-RMN, FTIR e análise da distribuição de peso molecular. Os perfis de gelificação foram avaliados com diagramas de fase, enquanto os mecanismos de gelificação e o comportamento viscoelástico foram estudados reologicamente. O desempenho das carrageninas híbridas numa formulação de gomas vegan também foi investigado, comparando-as com misturas comerciais e avaliando a influência de precursores biológicos, bem como a incorporação de um aditivo natural. A gelificação das KI foi mais dependente da concentração de polissacarídeo e da força iónica, enquanto as K+I gelificaram sob a maioria das condições. Em situações específicas, como altas concentrações de polissacarídeo em amostras ricas em ι ou equilíbrio κ/ι, e forças iónicas baixas em amostras ricas em κ, observou-se uma convergência nos perfis de gelificação. As K+I produziram géis mais rígidos e fortes, mas as KI apresentaram elasticidade superior em algumas condições, destacando o seu potencial em aplicações específicas. Contudo, maior elasticidade implicou menor resistência dos géis à deformação, resultando em maior fragilidade. Na formulação de gomas, as KI mostraram potencial como substitutos das K+I em termos de desempenho térmico, mas as misturas produziram géis mais fortes. O tratamento alcalino das KI aumentou a rigidez e a resistência à deformação dos géis, enquanto a incorporação do material biohíbrido melhorou a rigidez, mas reduziu a resistência, indicando a necessidade de otimização para equilibrar o desempenho técnico e a perceção do consumidor. Estes resultados evidenciam o potencial das carrageninas híbridas como alternativas a misturas comerciais, com aplicações promissoras em alimentos funcionais. Em última análise, a viabilidade das carrageninas híbridas como substitutos de misturas tradicionais depende do equilíbrio desejado entre força, elasticidade e resistência à deformação do gel. Palavras-chave: Carragenina híbrida, Misturas de carrageninas, Reologia, Carragenina K-2, Indústria alimentar vii ABSTRACT Hybrid Carrageenans as Alternatives to Commercial Blends: Rheological Properties and Performance in a Food Application This study provides a comprehensive comparison between the thermal-rheological properties of hybrid carrageenans (KI) and commercial K/I carrageenan blends (K+I) with equivalent κ content, focusing on hydrogels formed under varying salt and carrageenan concentrations. 1H-NMR, FTIR, and molecular weight distribution analyses were employed to characterize hybrid carrageenans, while phase diagrams and rheological tests (temperature, frequency, and strain sweeps) evaluated gel formation profiles, gelling mechanisms, and viscoelastic properties. Additionally, the study investigated the performance of hybrid carrageenans in vegan gummy candy formulations, comparing them to commercial blends and assessing the influence of biosynthetic precursors and the incorporation of a natural additive. KI showed greater dependence on polysaccharide concentration and ionic strength for gel formation compared to K+I, which gelled more readily under most conditions. KI demonstrated comparable performance under specific conditions, such as higher polysaccharide concentrations for ι-rich and balanced κ/ι samples or lower ionic strengths for κ-rich samples. While K+I generally produced stronger and stiffer gels, KI surpassed them in elasticity under certain ionic conditions, highlighting its potential for targeted applications despite trade-offs such as increased brittleness. In gummy candy formulations, KI successfully produced gel-like textures and demonstrated potential as a replacement for K+I in terms of thermal performance. While K+I produced stiffer and stronger gels, the alkaline treatment of hybrids generally enhanced their stiffness and strain resistance. The incorporation of the biohybrid material improved stiffness but reduced strain resistance, underscoring the need for formulation-specific optimization to balance technical performance and consumer appeal. These findings highlight the potential of hybrid carrageenans as clean-label, sustainable alternatives to commercial blends, with promising applications in functional foods. Ultimately, the viability of hybrid carrageenans as substitutes for commercial blends depends on the desired balance between gel strength, elasticity, and deformation resistance. Keywords: Hybrid carrageenan, Carrageenan blends, Rheology, K-2 carrageenan, Food industry xiv Blend 50κ50ι, with a balanced κ/ι ratio; and C) Sample J and Blend 90κ10ι, rich in κ-carrageenan. ........................................................................................................................................................ 86 Figure 19: Microscopic images of gummy candy samples using untreated carrageenan C (UT-C) (A, D), alkali-treated carrageenan C (T-C) (B, E), and commercial blend 10κ90ι (C, F), without (A-C) and with (D-F) the addition of a biohybrid material (BH). The biohybrid, composed of anthocyanins adsorbed onto bentonite, appears as red or green particles (D-F). ............................................................................ 87 Figure 20: FTIR-ATR spectra of gummy candies prepared, using untreated carrageenan (UT-J), alkali treated carrageenan (T-J) and the chemical equivalent commercial blend (90κ10ι), with and without the biohybrid material (BH), for sample J. ............................................................................................... 96 xv LIST OF TABLES Table 1: FTIR band assignment for each carrageenan disaccharide a ............................................... 31 Table 2: 1H-NMR chemical shifts (ppm) of the α-anomeric protons of carrageenan disaccharides b ... 32 Table 3: Formula of the gummy samples, expressed as weight percentage (% w/w) ......................... 47 Table 4: Synthesized description of the comparative systems studied, and labelling of vegan gummy candies produced ............................................................................................................................. 48 Table 5: Phase diagrams of KI (left) and of K+I blends (right), in KCl ............................................... 64 Table 6: Phase diagrams of KI (left) and of K+I blends (right), in NaCl ............................................. 67 Table 7: Values of Tg, Tstart1 and Tstart2, along with their associated errors for each sample, for both KCl and NaCl, as well as a characterization of the gelation mechanism (single-step or two-step process) ......... 71 Table 8: Rheological properties of hybrid carrageenans and commercial blends gelled in KCl and NaCl under varying polysaccharide and ionic conditions. The table includes gel elasticity in the LVE range (G0), limiting strain (γL), and flow strain (γF), as well as strain hardening and softening behaviors under large amplitude oscillatory shear (LAOS). Cases where strain softening is followed by strain hardening are indicated as “No then Yes” ............................................................................................................... 80 Table 9: Elastic and rheological properties of gummy candy samples, prepared using commercial carrageenan blends (mol.% κ; mol.% ι), KOH-treated (T-) or untreated (UT-) hybrid-carrageenans, with (+ BH) or without the biohybrid material. The table presents the gel elasticity in the LVE range (G0), limiting strain (γL), and flow point (γF), as well as strain hardening and softening behaviors under large amplitude oscillatory shear (LAOS). Cases where strain softening is followed by strain hardening are indicated as “No then Yes” .................................................................................................................................. 92 xvi LIST OF ABBREVIATIONS ACN AFM AG ATR BH CG CGS CS DRIFT DS DSC EFSA FDA FTIR GC GRAS IR KI K+I LAOS LVE NMR PDI PSS PSD SAOS SAXS SEC SEM SS Anthocyanin Atomic Force Microscopy Anhydrogalactose Attenuated Total Reflectance Biohybrid Material Clear Gel Clear Gel with Syneresis Clear Solution Diffuse Reflectance Infrared Fourier Transform Spectroscopy Degree of Substitution Differential Scanning Calorimetry European Food Safety Authority Food and Drug Administration Fourier Transform Infrared Spectroscopy Gummy Candy Generally Regarded as Safe Infrared Hybrid Carrageenan Commercial Carrageenan Blends Large Amplitude Oscillatory Shear Linear Viscoelastic (region) Nuclear Magnetic Resonance Polydispersity Index Phase-Separated (settled) Suspension Particle Size Distribution Small Amplitude Oscillatory Shear Small-Angle X-ray Scattering Size Exclusion Chromatography Scanning Electron Microscopy Stable Suspension xvii TG TGS TPA TS T-KI UT-KI XAN ZP Turbid Gel Turbid Gel with Syneresis Texture Profile Analysis Turbid Solution Treated Hybrid Carrageenans Untreated Hybrid Carrageenans Xanthan Gum Zeta Potential xix LIST OF SYMBOLS  ι λ µ ν K I L K-2 G’ G’’ G0 Mw γL γF Tg Tstart Kappa-carrageenan disaccharide moiety Iota-carrageenan disaccharide moiety Lambda-carrageenan disaccharide moiety Mu-carrageenan disaccharide moiety Nu-carrageenan disaccharide moiety Kappa-carrageenan polymer Iota-carrageenan polymer Lambda-carrageenan polymer Kappa-2 carrageenan, kappa/iota hybrid or weak-gelling kappa-carrageenan polymer Storage or elastic modulus (Pa) Loss or viscous modulus (Pa) Elastic modulus in the linear viscoelastic region (Pa) Weight-average molecular mass (g/mol) Limiting strain (%) Flow strain (%) Gelation temperature (ºC) Gelation onset temperature (ºC) 20 1. INTRODUCTION 1.1 Context and Motivation 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 (Blakemore, 2016; Souza et al., 2023). They play a crucial role in the formulation of a wide range of products, including dairy goods, meat products, and plant-based alternatives (Blakemore, 2016; Hotchkiss et al., 2016). The versatility and functionality of carrageenans make them indispensable in food technology, contributing to texture, mouthfeel, and product stability. The focus of most commercial applications has been on the use of K (kappa) and I (iota)- carrageenans, either in pure forms or as blends, to achieve desired textural properties. Extensive research has been conducted on these carrageenans, particularly in terms of their chemical structure, gelling mechanisms, and interactions with other food ingredients (de Vries, 2002; Souza et al., 2023). However, the increasing demand for K and I carrageenans in both food and non-food industries has recently exerted significant pressure on carrageenan production and seaweed farming (Azevedo et al., 2014; Bixler & Porse, 2011), making it urgent to look for underexploited and sustainable resources. In this context, K/I-hybrid carrageenans, which consist of a random block copolymer of κ-carrageenan and ι-carrageenan sequences, have emerged as promising alternatives to traditional commercial blends (Bixler et al., 2001; Villanueva et al., 2004). Industrially known as weak kappa or K-2, these hybrids form thermoreversible hydrogels with intermediate elasticity in comparison to those of K and I, offering unique structural and performance advantages in certain applications (Bixler et al., 2001; Hilliou, 2014; Hotchkiss et al., 2016; Villanueva et al., 2004). Moreover, they are predominantly sourced from wild seaweeds in cold waters, unlike K and I carrageenans, which are mainly obtained from intense farming of warm-water species, with associated production issues (Ward et al., 2020). This makes hybrid carrageenans not only a more sustainable option but also potentially more cost-effective, by eliminating the need for blending different types of carrageenans (Souza et al., 2023). Despite their promise, hybrid carrageenans 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 (Ficko-Blean et al., 2015). While these 21 variables affect the mechanical and structural characteristics of the resulting hydrogel, the relationship between the gelation mechanism and the structure of hybrid carrageenans remains complex and poorly understood (Souza et al., 2023; van de Velde, 2008). Additionally, while the properties of commercial K and I carrageenans are relatively well-documented, this is not the case for their blends for which controversial models are proposed (Bhattacharyya et al., 2024; Geonzon et al., 2023). Furthermore, comprehensive studies comparing the gelling mechanisms, phase diagrams, and rheological properties of hybrids with traditional blends are still lacking (Azevedo et al., 2014; Souza et al., 2023). Understanding the viscoelastic properties of hybrid carrageenan gels under large deformation regimes is also crucial, as these properties directly impact their functionality in industrial processes. Continued research in this area will help to define the conditions under which hybrid carrageenans can be most effectively used, potentially leading to new applications and improved product formulations. This research is driven by both the scientific need to fill these gaps and the practical need to address challenges within the food industry. By exploring hybrid carrageenans and systematically comparing them with traditional commercial blends, this study aims to determine whether they can offer distinct structural and functional advantages, such as enhanced stability, improved texture, or greater processing efficiency. Moreover, these findings potentially contribute to a deeper understanding of the relationships between chemical structure, gelling behavior and viscoelastic properties of hybrid carrageenans, under small and large deformation regimes. Additionally, by evaluating their performance in a specific food application, this research seeks to highlight whether hybrid carrageenans can provide concrete benefits over conventional blends. These insights could help shape future research directions, guide industry practices, and contribute to the development of innovative food products that meet evolving consumer demands. 1.2 Research Objectives The primary goal of this research is to perform a comprehensive comparison between the thermalrheological properties of hybrid carrageenans and commercial K and I carrageenan blends—ensuring equivalent κ content –, focusing on the hydrogels produced. By evaluating these two carrageenan systems, the study seeks to determine the similarities and differences in how they behave under different salt and carrageenan compositions. This will provide insights into how these systems form gels, their elasticity, viscosity, and overall performance in conditions that mimic real-world applications. 22 The study also aims to compare the performance of hybrid carrageenans in a specific food application – vegan gummy candies – with traditional commercial blends, critically assessing potential benefits and limitations of using hybrid carrageenans as a viable alternative. Additionally, hybrid carrageenans without alkaline treatment were also tested in the gummy formulations, to investigate the effects of biosynthetic precursors on its overall performance. Furthermore, the effects of incorporating a natural additive into the formulation was also evaluated across all carrageenan systems studied. 1.3 Document Outline This dissertation is structured as follows: Chapter 1: Introduction provides an overview of the research context, motivation, and main objectives, emphasizing its relevance to both the field and market demands. Chapter 2: State of the Art reviews the current state of research related to this study, covering key concepts and major contributions to the field, while also addressing ongoing challenges and knowledge gaps. More specifically, it provides insights into distinct types of carrageenans, their industrial applications, common extraction and characterization methods, gelling mechanisms, and rheological properties. Additionally, this chapter also provides an overview of their use in functional food formulations. Chapter 3: Methodology outlines the research design and methods used in this study. It covers material selection, hybrid carrageenan extraction and characterization, preparation of commercial blends, and the methods used to evaluate their gelling mechanisms and rheological properties. The chapter also details the formulation of carrageenan-based gummies, and their assessment protocol. Chapter 4: Results and Discussions present the study’s findings and a thorough analysis of their significance. In the first part, the characterization of the hybrid carrageenans extracted is discussed, followed by a systematic comparison of their rheological properties, and phase diagrams, with those of the corresponding commercial blends. In the second part, the overall performance of hybrid carrageenans and traditional commercial blends in the formulation of vegan gummy candies is assessed, highlighting potential advantages or disadvantages of using hybrid carrageenans in food industry applications. Additionally, the effects of biological carrageenan precursors in the hybrid chemical structure, and the impact of incorporating a natural additive in the formulation are also evaluated. Chapter 5: Conclusions and Future Work summarizes key insights, study limitations, and recommendations for future research. 23 2. STATE OF THE ART This chapter provides a comprehensive review of the existing literature and current advancements relevant to this study. 2.1 Carrageenans Carrageenans are a type of marine biopolymers synthesized by specific red algae from the class Rhodophyta (Hotchkiss et al., 2016; Souza et al., 2023), comprising up to 75% of the dry weight of the algae (Pacheco-Quito et al., 2020; Pereira, Critchley, et al., 2009). Their main function, within the cell wall of the marine algae, includes providing resistive action to oceanic stresses, such as osmose pressure and desiccation during low tides (Ficko-Blean et al., 2015; Souza et al., 2023). These hydrophilic polysaccharides have been well recognized for the ability of their solutions to form thermoreversible hydrogels, thereby making them useful for various industries as gelling, thickening, and stabilizing agents (Azevedo, Torres, et al., 2015; Necas & Bartosikova, 2013). Besides their functional properties, their bioactive attributes, affordability and relative abundance, make carrageenans suitable for food, cosmetic and pharmaceutical applications (Ahmed et al., 2014; De Jesus Raposo et al., 2015; Pacheco-Quito et al., 2020; Udo et al., 2023), and more recently bioremediation applications (Alshahrani et al., 2021; Hao et al., 2023; Hossain et al., 2024; G. Sharma et al., 2022). 2.1.1 Chemical Structure Carrageenans are natural complex polysaccharides classified as sulfated galactans (Ficko-Blean et al., 2015; Pacheco-Quito et al., 2020), due to the presence of 15% to 40% of ester-sulphate within their molecular structure (Ciancia et al., 2020; Necas & Bartosikova, 2013; Pacheco-Quito et al., 2020; Véliz et al., 2017). This sulfur content substantiates their acidic and anionic nature. The structure of these polysaccharides is characterized by linear backbones of alternating D-galactose and 3,6-anhydrogalactose (3,6-AG) units, linked by specific glycosidic bonds (α-1,3 and β-1,4). This forms a repeating sugar unit called carrabiose moiety (Ciancia et al., 2020; Ficko-Blean et al., 2015; Necas & Bartosikova, 2013; van de Velde, 2008). Carrageenophytes differ from agar-producing algae (agarophytes) because of the specific stereochemical arrangement of the bicyclic α-3,6-AG unit in the galactan chain, which is in the Lconfiguration in agar and in the D-configuration in carrageenans (Ciancia et al., 2020; Ficko-Blean et al., 2015). While the chemistry of sulfated galactans from red seaweeds is well-documented, the biosynthetic 30 After extraction, solid algal residues are separated from the K-2-rich solution through filtration or centrifugation. The resulting hybrid carrageenan is typically concentrated by precipitation with alcohol or KCl, followed by pressing to remove excess liquid (Hotchkiss et al., 2016; Souza et al., 2023). Precipitation with KCl is often preferred for recovering K-2 with enhanced gelling properties (a higher κ fraction) or for partially removing non-carrageenan components from certain carrageenophytes (Barahona et al., 2021; A. Kravchenko et al., 2023; A. O. Kravchenko et al., 2020; Perez Recalde et al., 2016). Alternatively, hybrid carrageenan can be recovered as a film or powder through water evaporation or freeze-drying of the K-2-rich solution (Souza et al., 2023). The purity of the final carrageenan depends on the separation and recovery methods employed. Based on earlier extraction studies on a set of hybrid carrageenophytes (Azevedo et al., 2013; Azevedo, Torres, et al., 2015; Hilliou, Larotonda, et al., 2006), extraction parameters, namely, algal content in extraction solvent, extraction duration and temperature, alga-carrageenan sol separation, and alkaline modification procedure were employed as described in Section 3. 2.3 Carrageenans Characterization Techniques The chemical structure of carrageenans can be assessed through several analytical techniques, with NMR spectroscopy and FTIR (Fourier Transform Infrared Spectroscopy) being particularly common (Pereira, Amado, et al., 2009; Souza et al., 2023; van de Velde, 2008). Vibrational spectroscopy methods like FTIR stand out as practical tools for qualitatively assessing the molecular structure and composition of materials (Pereira et al., 2003). FTIR is a fast and non-destructive technique, which measures the absorption of infrared radiation by a sample, requiring only a small amount of material for accurate measurements. The resulting spectrum gives detailed information about the molecular bonds and chemical groups present, as these correspond to specific wavelengths (GómezOrdóñez & Rupérez, 2011; Pereira, Amado, et al., 2009; Pereira et al., 2003). Recent research combining Fourier transform algorithms with attenuated total reflectance (ATR) techniques has further enhanced traditional infrared spectroscopy, by increasing the resolution of spectral data, simplifying sample preparation, and broadening the range of materials that can be analyzed (Gómez-Ordóñez & Rupérez, 2011; Pereira, Amado, et al., 2009). When analyzing the structure of carbohydrates, five key frequency regions are typically identified in IR spectra (4000–650 cm-1), as described by Mathlouthi & Koenig (1987): (1) 3600–2800 cm-1: OH and CH 31 stretching vibrations; (2) 1500–1200 cm-1: local symmetry region; (3) 1200–950 cm-1: CO stretching vibrations; (4) 950–700 cm-1: the anomeric region; and (5) below 700 cm-1: the skeletal region. For carrageenans, relevant FTIR absorption bands are observed around 2920, 1370, 1240, 930, 845, 820 and 805 cm-1, as registered in Table 1. These bands provide insights into their structure, on major carrageenan types but also minor components, such as μ and ν (Chopin et al., 1999; Gómez-Ordóñez & Rupérez, 2011; Pereira, Amado, et al., 2009). However, while FTIR is a powerful tool, it does have limitations. It offers only a semi-quantitative analysis and may struggle to clearly identify the composition of complex or heterogeneous carrageenan structures. This is because multiple chemical groups can contribute to the same absorption band, complicating interpretation (Chopin & Whalen, 1993; Rochas et al., 1986). Table 1: FTIR band assignment for each carrageenan disaccharide a Wavelength (cm-1) Bond(s)/group(s) Assignment Found in Disaccharides 2920 C-H of total sugar content κ, ι, μ, ν 1370, 1240–1260 S=O of sulphate esters κ, ι, μ, ν 930, 1070 (shoulder) C–O of 3,6-anhydrogalactose κ, ι 840–850 C–O–SO3 on C4 of galactose κ, ι, μ, ν 820, 825 (shoulder) C–O–SO3 on C2 of galactose ν 815–820, 867 (shoulder) C–O–SO3 on C6 of galactose μ, ν 800–805, 905 (shoulder) C–O–SO3 on C2 of 3,6-anhydrogalactose ι a Adapted from Gómez-Ordóñez & Rupérez (2011), Azevedo et al. (2013) and Pereira, Amado, et al. (2009) NMR spectroscopy, which relies on the resulting signals from the interaction between atomic nuclei and an external magnetic field, provides insights into the chemical environment, structure, and composition of samples (Dais & Spyros, 2012). Specifically, 13C-NMR and 1H-NMR target carbon-13 and proton atoms (hydrogen), respectively (Souza et al., 2023). Although solid-state ¹³C-NMR spectroscopy has shown potential for qualitative evaluation of carrageenans in seaweed, it remains underutilized in this context (Pereira, Amado, et al., 2009; Pereira et al., 2003; Souza et al., 2023). 1H-NMR spectroscopy allows for the quantification of various carrageenan types by analyzing the position and intensity of the α-anomeric hydrogen resonances from the repeating disaccharide units, typically found between 5.1 and 5.7 ppm (Campo et al., 2009). The 1H-NMR chemical shifts (ppm) of the α-anomeric protons of carrageenan disaccharides κ, ι, μ, and ν are registered in Table 2. More 32 importantly, proton NMR enables a quantitative assessment of the carrageenan chemical structures, as the peaks intensities relate to the molar content of the corresponding nuclei. Table 2: 1H-NMR chemical shifts (ppm) of the α-anomeric protons of carrageenan disaccharides b Carrageenan disaccharide Chemical shifts (ppm) κ 5.09 ι 5.29 μ 5.24 ν 5.50 b Adapted from Moraes & Hilliou (2024) and Van De Velde et al. (2004) 2.4 Gelling Mechanism – Coil-to-Helix Ion-Specific Transition The gelling mechanism of carrageenans is widely recognized in literature as a two-step process: a coil-tohelix transition upon cooling in the presence of salt, followed by the self-assembly of helices, into a threedimensional network (Campo et al., 2009; Hilliou, 2014, 2021; van de Velde, 2008). The exact nature of the helices and superstructures, and their relation to gel elasticity has long been debated, and several models have been proposed (Beaumont et al., 2021; Geonzon et al., 2020; Hilliou, 2021; Schefer et al., 2015). However, further studies are required to fully understand structure-elasticity correlations. Atomic force microscopy (AFM) has recently provided detailed images of primary coils, secondary helices, and larger aggregated network structures for Kand I-carrageenans in the presence of sodium, calcium and potassium salts (Diener et al., 2019; Schefer et al., 2015). As salt concentration increases, electrostatic repulsion between negatively charged sulfate groups is reduced, promoting the formation of intramolecular helical structures. Expectedly, K-carrageenan undergoes this transition at lower salt concentrations compared to I-carrageenan. In NaCl and CaCl2 environments, a progressive transition of polymer chains from random coils to helical conformations is observed, with K-carrageenan forming rodlike, stiffer helices and I-carrageenan forming curlier, more flexible helices. In contrast, KCl triggers further self-assembly of these helical polymer strands into supramolecular structures of higher complexity at high salt concentrations. In the presence of potassium, Schefer et al. (2015) suggested that K-carrageenan forms rigid, twisted dimers that aggregate into stiff superstrands, contributing to brittle gel networks. Meanwhile, I- 33 carrageenan forms flexible, supercoiled multi-filament strands, contributing to more elastic gels. Diener et al. (2019) imaged quaternary structures of stacked super-stranded helices connected by helical branching in K-carrageenan with K+, while I-carrageenan appeared as loosely intertwined single helices, similar to its behavior in NaCl. Thus, a critical reading of these results suggests that both strand rigidity and connectivity in the network are responsible for the different elastic properties of Kand I-carrageenan gels. The screening power of the divalent cation Ca2+ is higher, when compared to the monovalent Na+, for both Kand I-carrageenan, resulting in conformational transition at relative lower salt concentrations. In general, the order of effectiveness for inducing coil-to-helix transition for K-carrageenan is K+ ≈ Ca2+ > Na+, whereas for I-carrageenan Ca2+ > K+ > Na+ is followed (Schefer et al., 2015). AFM images of K-2 carrageenans helical assembly are mostly like K-carrageenan, though displaying more branching and a less rod-like morphology (Sokolova et al., 2013; Souza et al., 2023). The coil-tohelix transitions in K-2 are distinct for its K and I blocks, similar to the behavior seen in K+I mixtures, where both components undergo independent coil-to-helix transitions, followed by self-assembly into helical structures. These observations were confirmed in differential scanning calorimetry (DSC) studies performed on K-2 solutions in the presence of different salts (van de Velde et al., 2005) and in NaCl with different ionic strengths (Souza et al., 2011). Rheological data on K-2 solutions cooled in NaCl also showed a temperature dependence of two steps in both the shear storage (G’) and loss modulus (G”), suggesting the aggregation of two types of helical assemblies (Hilliou et al., 2014; Hilliou & Gonçalves, 2007; Souza et al., 2011; Torres et al., 2017). However, carrageenan concentration, counter ion type and ionic strength highly influence the two steps gelling mechanism of K-2 and its thermal hysteresis (greater in the presence of K+ (Chanvrier et al., 2004)). In general, Na+ promotes two-step gelation in K-2 solutions and favors the independent coil-to-helix transitions of I and K blocks, followed by the self-assembly of the helical structures. Conversely, in KCl or CaCl2 environments, a more straightforward single-step gelation is usually seen in the rheological profiles (Bahari et al., 2022; Cosenza et al., 2014; Hilliou, Larontonda, et al., 2006; Hilliou & Gonçalves, 2007; Hughes et al., 2023; Torres et al., 2018). In K+I mixtures, elasticity may arise either from microphase separation or interpenetrating networks of ιand κ-rich domains (Brenner et al., 2014; Du et al., 2016; Geonzon et al., 2019; Hu et al., 2016; Parker et al., 1993) or even from co-aggregation of K and I helices into complex superstructures, which could explain the larger gel elasticity of K+I compared to the sum of individual gel elasticities (Bui et al., 34 2019). Although the gelling process in K-2 resembles that of K+I mixtures, the nature of its self-assembly and super helical structures, and the origin of gel elasticity, remain poorly understood. Further comparative investigations on K-2 and K+I blends under varying carrageenan concentrations, salt types, and ionic strengths are essential as studies on blends nearly exclusively focused on equal contents of κ and ι. These studies should include in-situ structural characterization to better understand the hierarchical structures and sources of elasticity in these gels (Souza et al., 2023). Alternatively, a more comprehensive comparative study of phase diagrams and gel properties of K-2 and K+I blends, covering a wider range of κ and ι compositions than in Azevedo et al. (2014) and Torres, Azevedo, et al. (2016), could be performed, as proposed here. 2.5 Viscoelastic Properties of Hybrid Carrageenans Despite of K-2’s reported intermediate elastic properties between K and I carrageenan gels, their linear viscoelastic properties are far less studied in literature. In general, the mechanical spectra of K-2, measured by Small Amplitude Oscillatory Shear (SAOS), show qualitative similarities with K or I gels (Souza et al., 2023). K-2 gels exhibit near-frequency-independent elastic modulus (G') with no significant water syneresis (Azevedo et al., 2013, 2014; Azevedo, Torres, et al., 2015; Ponthier et al., 2020; Souza et al., 2011; Torres, Azevedo, et al., 2016; Torres, Chenlo, et al., 2016; Torres et al., 2017, 2018), contrasting with K gels, which tend to release water during liquid-to-gel transition, particularly in the presence of K+ (Souza et al., 2023). However, the linear viscoelastic properties of K-2 gels are also influenced by various factors, such as chemical composition, polysaccharide concentration, salt type and ionic strength. 2.5.1 Effect of K-2 Chemical Composition and Molecular Mass The elastic modulus of K-2 is significantly influenced by its chemical composition, especially the molar fraction of κ. Higher κ content correlates with stronger gels, especially in KCl solutions (Hughes et al., 2023; van de Velde et al., 2005). However, few studies focus on the relationship between K-2’s composition and its viscoelastic properties (Souza et al., 2023). In contrast, no clear correlation is observed in NaCl, likely due to variations in molecular mass (Mw). The size of the K-2 chain influences the elasticity of gels formed in NaCl, with longer chains leading to gels with lower G’ (Hilliou, Larontonda, et al., 2006; Souza et al., 2011; Torres, Chenlo, et al., 2016), while no clear trend is seen in KCl (van de 35 Velde et al., 2005). Therefore, more studies are needed to understand the impact of K-2’s chemical composition and molecular mass on the gel elastic properties, in NaCl and KCl. 2.5.2 Effect of Salt Type and Ionic Strength As already reported in Section 2.4, the type of salt and its ionic strength significantly impact the gelation behavior. The elastic modulus increases with ionic strength, and gels formed in KCl and CaCl2 are typically stronger than those in NaCl, with K-carrageenan evidencing greater cation specificity. Overall, K-2 exhibits cation-specific aggregation, especially for K-helices (Souza et al., 2023). A study by Torres et al. (2018) showed that stronger K-2 gels were obtained in CaCl₂, while NaCl produced the weakest. However, the range of polysaccharide concentrations and ionic strength for which a gel is produced is very different for each salt. This finding is evident in studies comparing phase diagrams for different hybrid carrageenans concentrations in different salt types and concentrations, such as Azevedo et al. (2014), Torres, Azevedo, et al. (2016) and Torres et al. (2018). These studies have also highlighted how those parameters affect the formation of clear or turbid gels. Turbid gels are often associated with the formation of particulate suspensions for K-2 having more than 50 mol.% κ, along with an increase in ionic strength. 2.5.3 Effect of Polysaccharide Concentration Similarly to what is reported in rheological properties of K and I gels, the elastic modulus of K-2 increases with polysaccharide concentration, following an exponential trend (Torres, Chenlo, et al., 2016), or a power law (Hilliou, 2021). Power laws with diverse exponent values have been suggested, due to the wide range of K-2 compositions and salt conditions used to prepare the gels (Hilliou, 2021; Hilliou & Gonçalves, 2007; Torres, Azevedo, et al., 2016). However, it is possible to infer an increase of power law exponent values (n) with higher ionic strength, suggesting a shift from worm-like to stiffer rod-like filament structures (Carrillo et al., 2013; Doi & Kuzuu, 1980; Jones & Marques, 1990; Meng & Terentjev, 2016). Gel turbidity also correlates with higher salt or κ content, leading to coarser structures in K-2 gels and larger n exponent values (Souza et al., 2023). 2.5.4 Viscoelastic Properties under Large Deformation The study of the properties of gels under large deformations are of great industrial relevance and can contribute valuable insights to understand the gel’s structure. In the linear viscoelastic regime, small 36 deformations result in a proportional response where properties like the shear modulus (which quantifies stiffness) remain constant. However, under large deformations, carrageenan gels deviate from predictable, linear responses, displaying instead complex nonlinear behaviors – its shear modulus changes, often hardening or softening depending on the filamentous network structure (Souza et al., 2023). Rotational rheometry applies simple shear (as opposed to compressive or indenting forces) to study how the material behaves under deformation. Simple shear ensures symmetric deformation, making it easier to measure and interpret stress - strain relationships (Souza et al., 2023). Theoretically, filamentous networks show strain hardening, which means the gel becomes stiffer when subjected to larger strain before it eventually ruptures, due to finite extensibility of the filaments – i.e . they are stretched between crosslinks in the network, and this stretching causes them to stiffen as they approach their breaking point (Carrillo et al., 2013; Doi & Kuzuu, 1980; Hilliou, 2021; Meng & Terentjev, 2016). Such stretching also supposes some degree of freedom between crosslinks, that is, pre-tension of filaments by the network is not that big (Meng & Terentjev, 2016). Experimental data from Large Amplitude Oscillatory Shear (LAOS) shows that K-2 gels, in KCl, undergo strain hardening, showing a relatively soft elasticity, with shear elastic modulus typically below 1 kPa (Azevedo et al., 2014; Bahari et al., 2022; Hilliou, 2021; Hilliou et al., 2009), when compared to K gels, which have much stiffer properties (up to 10 kPa). Strain softening, in which gels become less stiff under increased strain, was reported for certain K (Diener et al., 2019; Flores et al., 2017; Hilliou, 2021), I (Diener et al., 2019; Flores et al., 2017), and K+I blends (Flores et al., 2017), though the mechanisms behind this behavior remain unclear (Souza et al., 2023). 2.6 Hybrid Carrageenans – Market Trends and Application in Functional Foods Carrageenan is currently the leading seaweed-derived food hydrocolloid and accounts for the sixth largest share of the global hydrocolloids market (in terms of value), after guar gum, gelatin, xanthan gum, cellulose gum, and arabic gum (Hotchkiss et al., 2016; Zhang et al., 2023). The global carrageenan market was worth $850 million in 2022 and is expected to grow at an average yearly growth rate of 6.2%, reaching $1.55 billion by 2032 (Pulidindi & Ahuja, 2023). Carrageenans, with a molecular mass above 100 kDa (Necas & Bartosikova, 2013), are generally recognized as safe (GRAS) for consumption by both the FDA (Food and Drug Administration) and EFSA 37 (European Food Safety Authority), where they are classified as food additives E407 (refined) and E407a (semi-refined) (Rioux & Turgeon, 2015; Udo et al., 2023). Although toxicological assessments have confirmed that food-grade carrageenan has minimal physiological impacts, some studies suggest that degraded carrageenans may cause gastrointestinal health problems (Gotteland et al., 2020; Han et al., 2019; O’Sullivan et al., 2010; Shang et al., 2017; Sun et al., 2019), raising concerns about its safety (Bixler, 2017; Necas & Bartosikova, 2013). These controversies significantly influenced the carrageenan market, driving consumer demand towards perceived safer alternatives like agar, xanthan gum, and guar gum (Pulidindi & Ahuja, 2023). Beyond their functional applications as texturizing, stabilizing and gelling agents, hybrid carrageenans also exhibit various bioactive properties and unique attributes, making them valuable across a broad range of fields besides the food industry. These include applications in cosmetics and personal care products (Dubey & Dubey, 2020; Kim et al., 2018; Shafie et al., 2022), the pharmaceutical and biomedical industries (Calvo et al., 2019; De Jesus Raposo et al., 2015; Ha et al., 2022; Mehvari et al., 2024; Pacheco-Quito et al., 2020; Padhi et al., 2016; Pradhan & Ki, 2023), and also bioremediation techniques (Alshahrani et al., 2021; Hao et al., 2023; G. Sharma et al., 2022). 2.6.1 Bioactive, Nutritional, and Health-Promoting Properties Regarding functional food applications, carrageenans have been shown to promote health and prevent diseases by exhibiting antiviral, antibacterial, antioxidant, antitumoral, anti-inflammatory and immuneboosting effects (Bhuyan et al., 2023; Boulho et al., 2017; Dousip et al., 2014; Pradhan et al., 2022; Premarathna et al., 2024; Valado et al., 2019; W. Wang et al., 2011). Carrageenans also help improve gut health by acting as prebiotics, stimulating the growth of beneficial gut microbiota and increasing the production of short-chain fatty acids, such as acetate, propionate, and butyrate (Cherry et al., 2019; Kumari et al., 2023; Li et al., 2017; Liu et al., 2015). Moreover, the high level of dietary fiber in these biopolymers also adds to their nutritional value, helping with weight management and reducing the risk of cardiovascular disease due to their cholesterol-lowering effects (Dousip et al., 2014; Valado et al., 2019). In addition, carrageenans represent important plant-based alternatives to animal-derived ingredients like gelatin, dairy, and processed meats, aligning with current market trends (Blakemore, 2016; Hotchkiss et al., 2016; Karim & Bhat, 2008; Necas & Bartosikova, 2013; Palanisamy et al., 2018; Pereira, Critchley, et al., 2009; Rashmi & Mona, 2023; Russo Spena et al., 2024; Song et al., 2022; Udo et al., 2023). 38 In general, the latter described bioactive and functional properties of carrageenans (synthetized in Figure 3) make them excellent candidates for inclusion in functional foods and nutraceuticals (Kumari et al., 2023; Prasetyaningrum et al., 2019), providing texture while also promoting health benefits beyond basic nutrition. 2.6.2 Interaction with other Hydrocolloids and Ingredients Potential synergistic interactions between carrageenans and other hydrocolloids have been studied to modify the texture, stability, and gelation properties of food products (Hotchkiss et al., 2016; Song et al., 2022; Tunieva et al., 2021). In particular, when combined with xanthan gum, K-carrageenans form gels with improved viscoelastic properties, optimized texture, enhanced stability and reduced water syneresis (liquid separation from gels), allowing for the creation of high-quality vegan formulations that closely replicate the sensory properties of traditional gelatin-based products (Avallone et al., 2023; Balasubramanian et al., 2018; Brenner et al., 2015; Tunieva & Spiridonov, 2020; Xu et al., 2022). Traditional gummy candies (GC) have gel-like structures and are made using a combination of sugars, water, gelling agents (typically gelatin), and may also incorporate fruits, acids, aromas, and food colorants (Cappa et al., 2015; Ge et al., 2021; Mutlu et al., 2018; Teixeira-Lemos et al., 2021). Besides choosing vegan alternatives to animal-based gelatin, some other ways to align GC to current market trends and consumer demands, may comprise utilizing natural colorants, incorporating plant extracts, vitamins, or fruit derivatives, with bioactive and functional properties, and substituting sugar with other sweeteners, Figure 3: Functional and bioactive properties of hybrid carrageenans. Adapted from Pacheco-Quito et al. (2020) and Pradhan & Ki (2023). 39 such as honey or stevia (Mutlu et al., 2018; Palachum et al., 2023; Roudbari et al., 2024; Tarahi et al., 2023). These innovations aim to enhance the nutritional profile of a traditionally non-nutritious product, transforming it into a healthier option. This is particularly significant given the widespread consumption of jellies and gummies, especially among children and adolescents under 17 years old, which has been associated with a high incidence of obesity, tooth decay, and hyperglycemia (Grand View Research, 2024; Khawaja et al., 2019; Rippe & Angelopoulos, 2016; Tarahi et al., 2023). Carrageenan-based vegan gummy supplements have also been successfully used as a medium for delivering pharmaceuticals and/or nutritional bioactives, especially for children (Cheng et al., 2022; Qiu et al., 2024; Rashmi & Mona, 2023; Tarahi et al., 2023b). In that context, bioactive compounds such as anthocyanins (ACNs) have gained great attention as a natural food colorant and functional ingredient (Lourenço et al., 2019; Rodriguez-Amaya, 2019). These compounds are responsible for the red, purple and blue hues observed in various fruits, vegetables, flowers and grains and exhibit potent antioxidant, anti-inflammatory, anti-allergic, anti-diabetic, and anticarcinogenic activities (Rodriguez-Amaya, 2019; Shipp & Abdel-Aal, 2010). However, ACN activity is highly dependent on external factors such as light, pH, oxygen, temperature, and interactions with other ingredients, which limits their use in certain applications (Albuquerque et al., 2021; Cortez et al., 2017; Rodriguez-Amaya, 2019). To mitigate these limitations, stabilization techniques are commonly employed, including copigmentation, exclusion of O2, encapsulation/microencapsulation within biopolymers and adsorption onto nanoclays (Cavalcanti et al., 2011; Coelho Leandro et al., 2021; Cortez et al., 2017; Koop et al., 2024; Mahdavi et al., 2014; Rodriguez-Amaya, 2019). Thus, this work aimed to extract and characterize K/I hybrid carrageenans sourced from a diverse range of seaweed, focusing on their viscoelastic properties and their gelling behavior, under varying salt and carrageenan concentrations. These findings were systematically compared with commercial carrageenan blends of equivalent κ and ι composition. In response to current market trends for plant-based functional products, a hybrid carrageenan-based vegan gummy candy was developed and characterized. In this context, the hybrid carrageenan’s performance in this specific food application was compared to that of commercial carrageenan blends, of identical compositions. Moreover, the effects of including biosynthetic carrageenan precursors and the incorporation of an anthocyanin-based natural additive were determined by measuring the viscoelastic properties and overall stability of the formulation. 46 samples were chosen based on their distinct κ/ι ratios and gelling profiles, as they demonstrated gel formation across a broader range of salt and polysaccharide concentrations. For each sample, three different polysaccharide concentrations and ionic strengths were selected to provide a comprehensive understanding of their viscoelastic properties. 3.5.2 Rotational Rheometry Procedure The viscoelastic properties were measured using a stress-controlled rotational AR-G2 rheometer (TA Instruments Ltd, New Castle, DE, USA), equipped with a 25-mm parallel-plate geometry and a gap of 500 μm, at 85 °C. To prevent water evaporation during testing, dodecane (Sigma-Aldrich, St. Louis, MO, USA) was applied around the sample rim. Data acquisition was carried out using Rheology Advantage software (TA Instruments Ltd, New Castle, DE, USA). A time sweep was performed, where hot solutions were cooled from 85 °C to 25 °C, over 1.5 hours to assess the carrageenan sol-gel transition. The strain amplitude and frequency were set to 0.5% and 1.0 Hz, respectively, essentially to avoid any strain-induced change in the gel-setting behavior. During cooling, the rheometer’s gap was adjusted to account for the thermal expansion of the plates, while the normal force was maintained at 0 ± 1 N to compensate for potential volume changes during the sol-togel transition. Subsequently, the mechanical spectrum of the hydrogels at 25 °C was measured using a frequency sweep from 100 to 0.01 Hz, with a strain amplitude of 0.5%, essentially to keep the strain within the linear regime of viscoelasticity. Lastly, the linear and large deformation viscoelastic behavior of the hydrogels was analyzed at 25 °C by performing a strain amplitude sweep between 0.1% and 1000%, with a fixed frequency of 1 Hz. 3.6 Vegan Gummy Candy Formulation and Preparation 3.6.1 Raw materials and Formulation Inspired by a study conducted by Song et al. (2022), the vegan gummy candies (GC) formula included a mixture of carrageenans (KI or K+I), distilled water, xanthan gum (XAN, food grade), rosemary honey, and a natural additive (Table 3). Xanthan gum was used due to its documented synergistic effects when mixed 47 with K-carrageenan (Avallone et al., 2023; Balasubramanian et al., 2018; Brenner et al., 2015; Tunieva & Spiridonov, 2020; Xu et al., 2022), assuming similar behavior occurs with K/I hybrids. Rosemary honey served as a nutritious sweetener and texturizer, chosen for its therapeutic and preventive effects against inflammation-mediated chronic diseases (Ranneh et al., 2021). Rich in distinctive phenolic and flavonoid compounds, honey is reported to have health benefits, including antioxidant (Ranneh et al., 2018), anti-proliferative (Jaganathan et al., 2014), and anti-bacterial activity (Khan et al., 2018). The natural additive was a biohybrid material (BH) composed of anthocyanins (ACNs) extracted from jambolan fruit ( Syzygium cumini ), which were stabilized by adsorption onto bentonite. The BH was kindly provided by Professor Germán Valencia, of the Department of Chemical and Food Engineering of the Federal University of Santa Catarina, Campus João David Ferreira Lima (Florianópolis, Brazil). It was used due to its antioxidant and antimicrobial activities, attributed to anthocyanins, as well as its increased stability (Koop et al., 2024). For the formulation of the vegan gummy candies, three different alkali (KOH) treated hybrid carrageenans (T-KI) with significantly varying κ/ι ratios (samples C, F, and J) were evaluated, along with their respective commercial blends of similar chemical composition. Additionally, to investigate the potential effects of the carrageenan precursors μ and ν on the overall performance of the gummies, untreated hybrid carrageenans (UT-KI) extracted from the same sources (carrageenans C, F, and J) were also assessed. In addition, each GC formulation was replicated without the BH material to better understand its impact on the stability, texture, and structural profile of the final product. Table 3: Formula of the gummy samples, expressed as weight percentage (% w/w) Ingredients GC Samples with BH (% w/w) GC Samples without BH (% w/w) Water 86,5 87 Honey 10 10 Carrageenan (UT-KI, T-KI, or K+I) 2 2 Xanthan gum 1 1 Biohybrid 0,5 -------- 48 Therefore, a total of 18 GC samples were produced, considering three different κ/ι ratios tested in three different systems (UT-KI, T-KI, and K+I), each assessed with and without the biohybrid material, as shown in Table 4. Table 4: Synthesized description of the comparative systems studied, and labelling of vegan gummy candies produced Untreated Hybrid Carrageenans (UT-KI) Alkali-treated Hybrid Carrageenans (T-KI) Commercial Carrageenan Blends (K+I) GC Samples with BH UT-C + BH T-C + BH 10κ+90ι + BH UT-F + BH T-F + BH 50κ+50ι + BH UT-J + BH T-J + BH 90κ+10ι + BH GC Samples without BH UT-C T-C 10κ+90ι UT-F T-F 50κ+50ι UT-J T-J 90κ+10ι 3.6.2 Gummy Candies Preparation Procedure The preparation procedure of the gummies was inspired on Song et al. (2022). To prepare 10 g of GC samples, 0.2 g of carrageenan and 0.1 g of xanthan gum were weighed and gradually dispersed into a portion of distilled water, stirring vigorously with a spatula to prevent clumping. The solution was then heated to 80 °C with continuous magnetic stirring for 30 minutes. Meanwhile, the remaining ingredients (the rest of the water, honey, and the biohybrid, or just water and honey for the control groups) were weighed and mixed at room temperature until a homogenous solution was obtained. This mixture was added to the carrageenan and xanthan gum solution after the initial 30-minute heating period of the latter, and the resulting preparation was then heated and stirred for an additional 30 minutes. Finally, the samples were left to rest at room temperature overnight to promote gelation. After visually inspecting the phase state and coloration of the GC samples, they were stored in the fridge for preservation and taken out only for testing purposes. 49 3.6.3 Particle Size Distribution and Zeta Potential of the Biohybrid The particle size distribution (PSD) and zeta potential (ZP) of the biohybrid material dispersed in water were analyzed to evaluate the magnitude of the electrostatic charge on the particle surfaces and their size. These factors are crucial for understanding potential interactions with other ingredients in the formulation, as well as their impact on the product's overall stability and consistency (Parupudi et al., 2022). PSD was measured using a Mastersizer 3000 (Malvern Instruments Ltd., Worcestershire, UK) based on laser-light scattering principles. Due to equipment database limitations, the sample was approximated as silica (SiO₂), with a refractive index of 1.457 and an absorption index of 0.010. Water was used as the dispersant, with a refractive index of 1.330. The ZP was measured using the Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK), which uses Laser Doppler Velocimetry to determine electrophoretic mobility. Measurements were conducted in triplicate at 25 °C. 3.7 Gummy Candy Characterization 3.7.1 Optical Microscopic Imaging Optical microscopic imaging was used to inspect the quality of the dispersion of biohybrid particles along the formulation matrix, using an optical transmission microscopy (Olympus BH2, 20x) coupled to a LEICA DFC 280 digital camera. Melted samples (≈ 85 °C) were placed on microscope slabs and gently squeezed with a glass cover. 3.7.2 pH Measurements The pH of all samples was measured using a HI 2210 pH Meter (Hanna Instruments, Limena, Italy). 3.7.3 Rheological Characterization All GC samples were rheological characterized following the same methodology as described in Section 3.5.2. 50 3.7.4 FTIR-ATR Spectra Acquisition The FTIR-ATR analysis was conducted to understand whether the addition of the biohybrid material causes chemical changes within the matrix. For this assessment, a small sample of each gummy was collected, heated under reflux, at 80 °C, and finally dried overnight, to form a film. The FTIR-ATR spectra acquisition procedure used was the same as described in Section 3.3.1. 51 4. RESULTS AND DISCUSSION 4.1 Chemical Structure of Hybrid Carrageenans Insights on the chemical characterization of the hybrid carrageenans produced were mainly obtained by FTIR-ATR, 1H-NMR and SEC, to identify the material's molecular bonds and chemical groups, chemical composition and molecular mass distribution, respectively. 4.1.1 FTIR-ATR Spectra All carrageenan samples, both KOHand NaOH-treated, were analyzed using FTIR-ATR. However, since they exhibited similar spectral behaviors, only the spectra obtained from the KOH-treated samples are presented and discussed here. Thus, Figure 7 shows the FTIR-ATR spectra of the KOH-treated carrageenan samples within the range of 1400–600 cm−1. The FTIR-ATR spectra of the NaOH-treated carrageenan samples are registered in Figure A.1, in Appendix A.1 for further consultation. The FTIR spectra of the sample films revealed distinct carrageenan profiles. In particular, samples A, B, C and D displayed a more pronounced band at 805 cm-1 and a shoulder around 905 cm-1, both associated to 3,6-anhydro-D-galactose-2-sulphate, characteristic of ι-carrageenan disaccharide units. Figure 7: FTIR-ATR spectra of hybrid carrageenan samples treated with KOH, highlighting the most characteristic bands of carrageenans at 1240, 930, 845, and 805 cm⁻¹. Note: The FTIR-ATR spectra for samples F and N, in KOH, are not shown due to poor resolution. 52 Conversely, samples I, J, K, L, M and O exhibited a prominent band near 930 cm-1, alongside a shoulder at 1070 cm-1, which are assigned to 3,6-anydro-D-galactose moieties typically found in ι, but also κ-carrageenan units. Samples E, G and H presented a spectrum with broader and less well-defined peaks around key wavelengths ( e.g. , 930 cm-1 and 805 cm-1), implying weaker structural contrasts. This profile may correlate with a more balanced or homogeneous distribution of κand ι-carrageenan fractions, leading to an overall blending of vibrational modes, in which the distinctive features of pure κor ι-carrageenan would be less pronounced. In addition, the FTIR-ATR spectra for samples F and N, in KOH, were not presented due to poor resolution, likely caused by residues or impurities in the film which may have hindered an accurate assessment. Common to all carrageenan samples, one band appeared at approximately 2920 cm−1 on the FTIR spectra (data not shown) assigned to C–H stretching vibrations, corresponding to the total sugar content. Moreover, all samples showed bands at approximately 845 and 1240 cm-1, corresponding to d-galactose4-sulphate and sulphate ester groups, respectively, which are found across all carrabiose types (κ, ι, μ and ν). However, bands associated with μand ν-carrageenan, typically near 820 cm-1, were not identified, suggesting that these types are either present in lower concentrations or their structural features are less detectable under the current conditions. Ratio of Absorbances at 1370/2920 cm1 – Sulfate-to-Sugar Index The peak absorbances associated with total sulfate groups at 1240 cm-1 and 1370 cm-1 can be normalized using the peak corresponding to C-H stretching vibrations at 2920 cm-1, which represents the total sugar content. The resulting ratios, A1240/A2920 and A1370/A2920, serve as semi-quantitative indicators of the sulfate content, expressed as the degree of substitution (DS)—the average number of sulfate groups (in moles) per disaccharide unit in carrageenan (Azevedo et al., 2013; Rochas et al., 1986). According to Rochas et al. (1986), the accuracy of sulfate content estimation using these ratios is reliable for polymers with a DS of less than 0.8 for A1240/A2920 and more than 0.4 for A1370/A2920, making the latter appropriate for determining sulfate content across a broader range of sulfated galactans. The acuity of the DS determination with the A1370/A2920 ratio was recently verified with the FTIR analysis of films made of 4 blends of κ and ι with different ratios, spanning theoretical DS from 1 to 2 whereas the band ratio ranged from 1.3 to 1.9 (de Oliveira et al., 2020). 53 Theoretically, κ-carrageenan contains, on average, one sulfate group per disaccharide unit, while ιand μ-carrageenans each contain two sulfate groups, and ν-carrageenan carries three sulfate groups per disaccharide (Rochas et al., 1986; van de Velde, 2008). Assuming that the hybrid carrageenans produced consist only of these four components, each contributing varying sulfate groups, it is expected that the DS sulfate values would ideally be above 1. Therefore, the ratio A1370/A2920 was used as a semiquantitative index relating total sulfate to sugar content, as represented in Figure 8, for samples treated with KOH and NaOH. As all ratios in Figure 8 are above 2, a DS cannot be extracted from the data using the calibration carried out elsewhere (de Oliveira et al., 2020). Thus, the present analysis of the sulphate content is only qualitative. According to Figure 8, the samples exhibited varying degrees of sulfation, with NaOH-treated samples generally showing higher sulfate-to-sugar ratios compared to KOH-treated ones, especially for samples B, E, G, H, M, N, and O. This suggests that NaOH-treated samples tend to preserve sulfation better than KOH-treated samples, which could impact their gelation, viscosity, and overall performance. Some samples (A, I, J, K, and L) had similar ratios between treatments, indicating less difference in sulfate retention. Samples I, J, K, and L showed consistently lower A1370/A2920 ratios, possibly due to a higher content of less sulfated moieties, such as κ-carrageenan. Several factors beyond the actual presence of sulfated carrageenan fractions can contribute to higher sulfate content in hybrid carrageenan extracts. Co-extracted materials such as floridean starch, proteins, Figure 8: Ratio of absorbance at 1370/2920 cm-1 (A1370/A2920), indicative of the total sulfate content in carrageenan samples treated with KOH (light grey) and NaOH (dark grey). Note: the standard deviation for each ratio falls within the range of 10 -3 to 10-4. While it has been included in the bar chart, it is not prominently visible due to its relatively small magnitude. 54 or residual sulfate salts (due to the absence of pre-washing) may lead to misleading estimations of sulfate levels (Moses et al., 2015). Additionally, ion pair formation between sulfate groups and counter-ions (Na⁺ or K⁺) can enhance hydration, alter the vibrational energy, and increase absorbance at 1370 cm-1 (Rochas et al., 1986). This interaction could result in a higher A1370/A2920 ratio, reflecting ionic influences on FTIR readings. Ratio of Absorbances at 805/845 cm-1 - Relative Amount of ι-carrageenan Additionally, the absorbance of the 805 cm-1 peak (assigned to 3,6-anhydro-D-galactose-2-sulfate) was normalized with respect to the 845 cm-1 peak (assigned to D-galactose-4-sulfate), with the A805/A845 ratio reflecting the relative amount of ι-carrageenan (Azevedo et al., 2013). The A805/A845 ratio is presented in Figure 9, for both KOH and NaOH-treated samples. The ratio A805/A845 suggests that the NaOH and KOH treatments yielded chemically distinct carrageenans, with KOH-treated carrageenans (light grey) consistently showing a slightly higher relative ι-carrageenan content, especially in samples F, G, H, J, M, N, and O. In both scenarios, sample N presented the highest A805/A845 ratio. A possible explanation to the higher A805/A845 ratios observed in KOH-treated samples and, particularly, in sample N could be the presence of higher concentrations of precursor units in the carrageenan chain. The absorption bands between 815 and 825 cm-1, assigned to the sulfate groups on Figure 9: Ratio of absorbance at 805/845 cm-1 (A805/A845), indicative of the relative amount of ι-carrageenan in samples treated with KOH (light grey) and NaOH (dark grey). Note: the standard deviation for each ratio falls within the range of 10⁻³. While it has been included in the bar chart, it is not prominently visible due to its relatively small magnitude. 55 C2 and C6 of galactose found in carrageenans μ and ν, could affect the intensity of the band at 805 cm-1 attributed to the sulfate group on C2 of 3,6-anhydrogalactose, leading to an overestimation of the relative ι-carrageenan content. According to Figure 9, in NaOH, samples A, B, C, D, and N exhibited a higher A805/A845 ratio, indicative of greater ι-carrageenan content, followed by samples E, F, G, H, O, and M. Samples I, J, K, and L showed the lowest relative ι-carrageenan content in both alkaline solutions. 4.1.2 1H-NMR FTIR spectroscopy data were confirmed by NMR spectroscopy analysis, as the chemical composition of all samples were analyzed using 1H-NMR. Figure 10 presents the ¹H-NMR spectra of three chemically representative samples, processed with NMRium® software, along with the chemical shifts corresponding to the anomeric protons of the primary carrabiose units (κ, ι, μ, and ν). The molar fractions (mol.%) of each carrageenan disaccharide repeating unit are shown in Figures 11A and 11B for KOHand NaOHtreated samples, respectively. The 1H-NMR spectra revealed slight deviations from the predicted chemical shifts for each anomeric proton of the κ, ι, μ, and ν carrabiose units (Table 2), with κ-carrageenan peaks appearing between 5.07 and 5.11 ppm, ι-carrageenan around 5.27 to 5.31 ppm, μ-carrageenan between 5.19 and 5.21 ppm, and ν-carrageenan from 5.46 to 5.51 ppm. Figure 10: 1H-NMR spectra of three significantly different KOH-treated samples, in terms of chemical composition, obtained via NMRium® software: sample B, rich in ι-carrageenan; sample F, with a more balanced κ/ι ratio, and sample I, rich in κ-carrageenan. Vertical dotted lines indicate the peaks of the assigned disaccharide units showing up at chemical shifts referenced in Van De Velde et al. (2004). 62 in balanced κ/ι systems might weaken inter-chain interactions and loosen network structures. Additionally, other potential factors influencing molecular mass and chemical composition relationships should be explored to provide a more comprehensive understanding of these mechanisms. Upon comparing the molecular mass data of the untreated samples with those from Figures 12A and 12B, it is evident that alkaline treatment generally led to a reduction in molecular mass, with a few exceptions—specifically, samples G and L in KOH, and C’, D’, G’, and L’ in NaOH. Alkaline treatment, particularly at elevated temperatures, can cleave the glycosidic bonds within carrageenan chains. This process, known as alkaline hydrolysis, breaks the polymer into smaller fragments, resulting in lower molecular mass values (Fuchs et al., 2024). The extent of this effect depends heavily on treatment conditions (temperature, pH, and duration), with harsher conditions inducing more significant chain scission, especially in NaOH solutions (Azevedo et al., 2013; Azevedo, Torres, et al., 2015). However, the decrease in molecular mass was more prominent in most KOH-treated samples, with exceptions such as samples H and I, which exhibited lower Mw values in NaOH, and sample A, which showed a similar decrease in both alkaline solutions. 4.2 Hybrid Carrageenans vs Commercial Blends Selected hybrid carrageenans produced were systematically compared with commercial carrageenan blends of equivalent chemical composition through phase diagrams, in NaCl and KCl, for samples treated with NaOH and KOH, respectively. Subsequently, the rheological properties of selected gels were evaluated using rotational rheometry under small and large deformation. 4.2.1 Phase Diagrams of KI vs K+I in NaCl and KCl The phase diagrams of the selected hybrid carrageenans (KI), alongside the chemically equivalent commercial blends (K+I) at comparable salt and polysaccharide concentrations, are presented in Table 5 for KCl and in Table 6 for NaCl. These samples were selected based on the criteria outlined in Section 3.4.1, ensuring a broad range of chemical compositions with minimal to no biological precursor content or interfering components, such as floridean starch. As an exception, sample E' (NaOH-treated), was intentionally included to assess potential gelling behavior differences due to starch content, as previously mentioned in Section 4.1.2. 63 The influence of salt type on the gelling behavior of Kand I-carrageenans is well documented: Kcarrageenan is more salt-specific, showing higher gelling efficiency in the presence of K⁺ ions. In contrast, I-carrageenan can also gel with Na⁺ ions, though the process is slower, producing softer, more elastic gels. Overall, K⁺ promotes faster and firmer gelation in K-carrageenan, while Na⁺ induces softer, more flexible gels, especially for I-carrageenan (Diener et al., 2019; Piculell, 2006; Schefer et al., 2015). In contrast, the salt dependency of K-2 carrageenan is not as well understood as pure Kor I-carrageenan. Overall, K-2 exhibits intermediate gelling behavior between the two types of carrageenans, showing cationspecific aggregation, especially for K-helices (Souza et al., 2023). However, the range of polysaccharide concentrations and ionic strength for which a gel is produced is very different for each salt (Azevedo et al., 2014; Torres, Azevedo, et al., 2016; Torres et al., 2018). Therefore, the main purpose of this section is to directly compare the gelling behavior of hybrid carrageenans with their commercial blend counterparts, along a wide range of compositions, in order to determine similarities and differences in how they behave under different salt and carrageenan compositions. Secondarily, the comparison between NaOH and KOH systems mainly intends to evaluate how K-2 hybrid carrageenans behave in the presence of different salts: K+ and Na+, since there is still a lack in the literature on this topic. Overall, the comparison of phase diagrams in the right and left columns confirmed notable differences between K/I-hybrid carrageenans and the commercial Kand I-carrageenan blends (Azevedo et al., 2014; Villanueva et al., 2004). Gel formation (in green) in the presence of K+ occurred more readily in commercial K+I blends than in KI carrageenans, happening across almost all chemical compositions (in terms of κ and ι content), polysaccharide concentrations, and ionic strengths, whereas KI carrageenans showed higher sensitivity to those parameters. For ι-rich samples (B and C), gelation was more dependent on the polysaccharide concentration, occurring only at KI ≥ 1 wt.%, indicating a threshold for forming a stable network. Below this concentration, KI samples formed stable suspensions, suggesting that while the polysaccharide chains are present, they are insufficient to form a fully connected gel. Instead, they may create microdomains or partial networks that do not completely immobilize the solvent, resulting in a suspension state rather than a solid gel. Given the high charge density of ι-carrageenan, 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 (Piculell, 2006). In contrast, all K+I samples formed gels across similar conditions. 64 κ-rich KI samples, particularly sample K (95 mol.% κ), behaved comparably to their commercial counterparts. Generally, for samples with a high κ composition, ionic strength played a more significant role, with gel formation particularly challenged at elevated K⁺ concentrations (≥ 0.5 mol/L for samples I, J, and K, and 1 mol/L for the K+I blend). While potassium ions more effectively induce the helical and aggregated states of K-carrageenan, above the optimal salt concentration solvation and/or salt-out effects may lead to precipitation, compromising gel formation. Similar high salt sensitivity was observed in samples E and M (60-70 mol.% κ) at K⁺ concentrations of 1 mol/L. These results align with other studies, which reported the formation of stable particle suspensions across all polymer concentrations at [K⁺] = 1 mol/L for KI samples with 75 mol.% κ (Torres, Azevedo, et al., 2016) and 70 mol.% κ (Azevedo et al., 2014). However, samples E and M gelation were also hindered by low ionic strengths and low polysaccharide concentrations. This observation contrasts with the previously mentioned studies, where samples formed clear or turbid gels under similar conditions (Azevedo et al., 2014; Torres, Azevedo, et al., 2016) in harmony with the trend found for κ-rich KI samples. 0.01 0.1 0.5 1 0.01 0.1 0.5 1 0.5 SS SS SS SS 1SS TG TG TG 2TG TG TG TG 0.5 SS SS SS SS 1SS TG SS TG 2TG TG TG TG 0.5 PSS SS SS SS 0.5 CG CGS CG TS 1 PSS SS PSS TS 1CG CGS TG TG 2TS TGS TGS TS 2CG CG TG TG 0.5 CS TG TGS TS 0.5 CG CGS TS TS 1 PSS TGS TGS TS 1CG CGS TG TG 2 TGS TGS TGS TS 2CG CGS TG TG 0.5 SS TG CG TS 0.5 CG CGS TS TS 1 TGS TGS TG TS 1 CGS CGS TG TG 2 TGS TG TGS SS 2CG CGS TGS TG 0.5 CGS CGS SS SS 1 TGS TGS TGS SS 2 TGS TGS SS PSS 0.5 CGS CGS TS PSS 1 TGS TGS SS PSS 2 TGS TG SS TS 0.5 CGS TGS TGS SS 1 TGS TGS TS PSS 2 TGS TGS TG PSS CS Clear Solution SS Stable Suspension CG Clear Gel CGS Clear Gel with Syneresis TS Turbid Solution PSS Phase Separated (setlled) Suspension TG Turbid Gel TGS Turbid Gel with Syneresis Phase Diagram in KCl CG TG TG CGS TGS TS J (87 κ + 13 ι) 1 CGS CGS TG TS M (69 κ + 31 ι) 70 κ + 30 ι I (90 κ + 10 ι) 90 κ + 10 ι 0.5 CG K (95 κ + 5 ι) 2 CGS TG TG F (48 κ + 47 ι) 50 κ + 50 ι E (56 κ + 44 ι) 60 κ + 40 ι TG 1 CG CG TG TG B (4 κ + 96 ι) 10 κ + 90 ι 0.5 CG CG CG C (6 κ + 91 ι) 2 CG CG KI Sample (%.mol κ + %.mol ι) KI (wt%) [K+] (mol/L) K+I Sample (%.mol κ + %.mol ι) K+I (wt%) [K+] (mol/L) Table 5: Phase diagrams of KI (left) and of K+I blends (right), in KCl 65 Sample F (K-2 carrageenan with ≈50 mol.% κ and 50 mol.% ι) exhibited the most difficulty in forming gels, which only occurred at high polysaccharide concentrations (2 wt% KI) and ionic strengths of 0.1-0.5 mol/L, in evident contrast to its K+I counterpart. Interestingly, while sample F (48 mol.% κ + 47 mol.% ι) differs from sample E (56 mol.% κ + 44 mol.% ι) in chemical composition, the extent of this difference is comparable to that between samples E and M (69 mol.% κ + 31 mol.% ι), yet the latter two exhibit similar gelling behaviors. This suggests that factors beyond κ and ι composition are influencing gelation. One significant factor could be the high polydispersity index (PDI) of sample F, coupled with its average molecular mass being close to the lower limit (Figure 12A), potentially resulting in a substantial proportion of chains too short to form gels. This could also account for the differences observed with its commercial blend counterpart, as commercial Kand I-carrageenans have significantly higher molecular masses (Figure B.2 – Annex B.2). Additionally, the relatively higher content of precursor units (≈ 5 mol.%) in sample F (Figure 11A) may hinder its ability to establish the robust network structures necessary for gelation under broader salt and polysaccharide conditions. Torres, Azevedo, et al. (2016) reported a K-2 carrageenan with a similar chemical composition to sample F (53 mol.% κ and 47 mol.% ι), extracted from A. devoniensis , that formed gels without syneresis for all conditions, except for 0.5-1 KI wt.%, at 0.01 mol/ [K+] which yielded turbid solutions. These differences suggest that intrinsic species-specific factors, such as K-2 chain length distributions and block length variability, also influence the overall gelling performance of extracted KI carrageenans, as the molecular masses of sample F and extract from A. devoniensis are both in the lower limit. This inherent variability, common to natural products, complicates straightforward correlations between K-2 hybrids' gelling properties and their κ or ι fractions. Exploring diverse algae sources could help clarify this variability and establish a clearer κ content threshold, above which KI hybrids behave more like commercial mixtures in terms of gelling ability. However, it is worth noting that both commercial carrageenans exhibit higher molecular masses than sample F, further complicating direct comparisons. Syneresis (depicted in light and dark fluorescent green) increased with higher κ-content (unnoticed for κ < 50 mol.%), especially in KI samples, where water separation occurred in a great part of gels. In commercial blends, syneresis was more common at 0.1 mol/L K⁺ for samples with ≥ 50 mol.% κ. These findings did not entirely align with a study by Azevedo et al. (2014), who observed no syneresis in KI gels with 70 mol.% κ in KCl, while K+I gels exhibited water release at higher K⁺ concentrations. In this study, syneresis occurred in both KI and K+I gels at [K⁺] < 1 mol/L. Both studies agree on the difficulty of gel 66 formation in κ-rich KI systems at 1 mol/L KCl. The discrepancies may result from differences in seaweed sampling (location and harvest time) and treatment methodology, as Azevedo et al. (2014) applied alkali treatment directly to seaweed pre-extraction, whereas this study treated the extracted carrageenan. This suggests that possible different Mw distributions, as well as κ and  distributions along the polysaccharide chain, give different gel syneresis, whereas the chemical structure is essentially identical in terms of κ and  contents. Note also that differences in the phase diagrams of K+I gels also point to possible differences in the experimental protocols employed (in particular the thermal history during cooling), in the assessment of phases (subjected to experimentalists criteria) or the K and I used, as commercial samples were sourced from the same provider. Clear gels (with or without syneresis) were more frequently observed in the commercial blend systems, particularly at low ionic strengths ([K+] ≤ 0.1 mol/L). This observation aligns with findings by Azevedo et al. (2014), who linked the transition from clear to turbid gels to changes in gel structure and a shift in the concentration dependence of critical strain for gel break up. In contrast, nearly all KI samples showed some degree of turbidity, with clear solutions appearing only occasionally at lower polysaccharide concentrations. The inherent tendency of KI to exhibit turbidity likely results from several factors: KI samples were extracted from unprocessed and unwashed algae, allowing for possible co-extraction of soluble and insoluble impurities that contribute to turbidity, whereas the commercial carrageenans in K+I blends were obtained in a highly processed, purified state. Leaving aside the optical role of impurities, the turbidity suggests that KI samples tend to yield more heterogeneous structures, potentially forming larger, irregular microdomains compared to commercial blends. This structural heterogeneity increases light scattering and turbidity, especially at higher polymer concentrations. Similarly to what was seen in KCl, in the presence of Na+ hybrid carrageenans also demonstrated a more complex and variable gelling behavior compared to the commercial blends (Table 6). In NaCl, gel formation for K+I samples was mostly conditioned by low polysaccharide concentrations, not showing much dependence on the ionic strength (contrarily to what is seen in KCl). Notably, commercial blends in NaCl produced clearer gels and solutions compared to KCl. As expected, due to the non-specific ion response of ι-carrageenan, ι-rich 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 K-2 carrageenans (samples F’, E’, H’ and M’) with ≈50-70 mol.% κ, no gels formed under any condition, 67 contrasting starkly with K+I counterparts. The study by Torres, Azevedo, et al. (2016), though aligning with the notable gelling difficulty of hybrid carrageenans with 50-50 mol.% and 75-25 mol.% of κ-ι, stated the formation of gels at 1-2 wt.% of polysaccharide and 0.5-1 mol/L [Na+]. Similarly, Azevedo et al. (2014) documented these results for 70-30 mol.% κ-ι hybrids. For samples E' (containing starch) and H', both with similar κ and ι fractions, the phase diagram analysis revealed no significant difference, suggesting that starch presence did not have a noticeable impact. However, due to the inherent gelling difficulty of K-2 hybrids in NaCl, it is challenging to definitively conclude that starch has no effect on gelling properties. Interestingly, some κ-rich gels registered the occurrence of syneresis in NaCl, which is not commonly reported in the literature. This happened specifically in the K+I blend with 70 %.mol κ, and for samples I’, J’ and K’, at higher polymer and Na+ concentrations. Although potassium ions are known to encourage syneresis in K-carrageenan gels due to their strong binding affinity, sodium ions generally have weaker interactions, so syneresis is less likely. Notably, in the case of the K+I blend with 70 mol.% κ, the results 0.01 0.1 0.5 1 0.01 0.1 0.5 1 0.5 SS CS PSS SS 1 PSS TG TG TG 2TG TG TG TG 0.5 SS SS SS SS 1SS TG TG TG 2TG TG TG TG 0.5 SS SS SS SS 0.5 CS CS CS CS 1 PSS PSS SS SS 1CG CG CG CG 2 PSS PSS TS TS 2CG CG CG TG 0.5 PSS PSS PSS PSS 1 PSS PSS PSS TS 2 PSS PSS PSS TS 0.5 CS SS PSS TS 1SS SS PSS TS 2SS SS TS TS 0.5 CS SS SS SS 0.5 CS CGS CG CGS 1 PSS SS SS SS 1CG CGS CGS TGS 2 PSS TS SS SS 2CG CGS CG TGS 0.5 CS PSS TS TS 1SS TG TGS TS 2TG TG TGS TGS 0.5 PSS SS PSS SS 1 PSS SS PSS PSS 2TG TG TG TGS 0.5 PSS PSS PSS PSS 1 PSS TG TGS PSS 2TG TG TG TGS CS Clear Solution SS Stable Suspension CG Clear Gel CGS Clear Gel with Syneresis TS Turbid Solution PSS Phase Separated (setlled) Suspension TG Turbid Gel TGS Turbid Gel with Syneresis [Na+] (mol/L) KI Sample (%.mol κ + %.mol ι) KI (wt%) [Na+] (mol/L) K+I Sample (%.mol κ + %.mol ι) K+I (wt%) C' (5 κ + 95 ι) 10 κ + 90 ι 0.5 CG CG D' (10 κ + 88 ι) 2 CG CG CG 1 CG CG CG TG CG CG CG TG M' (67 κ + 29 ι) 70 κ + 30 ι I' (90 κ + 10 ι) 90 κ + 10 ι 0.5 CS K' (93 κ + 7 ι) 2 CG Phase Diagram in NaCl 60 κ + 40 ι 0.5 2 CG CG CG TG CG CG F' (54 κ + 45 ι) 50 κ + 50 ι H' (62 κ + 37 ι) E' (57 κ + 42 ι) CG 1 CG CG CG CS CS CG CS CG CG TG CS CS CS J' (88 κ + 12 ι) 1 CG Table 6: Phase diagrams of KI (left) and of K+I blends (right), in NaCl 68 are even more unusual, as other studies using the same experimental protocol and source of commercial carrageenans did not report any syneresis (Azevedo et al., 2014; Torres, Azevedo, et al., 2016). This further supports the possibility of user-related experimental errors, although none could be specifically identified. One potential explanation could be that, during cooling, water vapor from the gel or solution may condense on the cooler walls of the flask, forming a liquid film that could be mistakenly identified as syneresis. To summarize this section, phase diagrams highlighted key differences between K/I hybrid carrageenans (KI) and commercial blends (K+I). KI carrageenans showed greater dependency on polysaccharide concentration and ionic strength for gel formation compared to K+I. Nonetheless, hybrid carrageenans exhibited gelation behaviors similar to commercial blends under specific conditions. These included higher polysaccharide concentrations for ι-rich samples in both KCl and NaCl, balanced κ/ι samples in KCl, and κ-rich samples in NaCl, as well as lower ionic strengths for κ-rich samples in KCl. However, hybrid carrageenans consistently formed more turbid gels. In comparing KI samples, KCl facilitated more consistent gel formation across concentrations, while NaCl resulted in diverse gelling behaviors, including more suspensions and phase separations. Samples rich in κ-carrageenan showed more specificity towards KCl, whereas ι-rich samples presented similar phase diagrams in NaCl and KCl. K-2 carrageenans displayed salt-specific responses, forming gels in KCl but not in NaCl, underscoring their limited gelling potential in Na+ environments, consistent with Azevedo et al. (2014). In conclusion, KI carrageenans may serve as viable alternatives to K+I under specific polysaccharide concentrations and ionic strengths conducive to gel formation in both. However, rheological testing is essential to evaluate their comparative elasticity, modulus, and yield stress for optimal application. Additionally, KI may find unique value as a stabilizer in non-gelling applications, such as sauces, dressings, or emulsions, where gel-like networks aren’t essential but water-binding and stable suspension properties are advantageous. 4.2.2 Rheological Characterization of Hydrogels In the rheological assessment, specific samples were selected to provide insight into gel behavior across varying conditions, given limited time for analysis. Gels from samples C, E, and K in KOH and C' and K' in NaOH (sample E’ did not form gels) were chosen, as these differed notably in κand ι-carrageenan 69 content, allowing the investigation of rheological responses tied to ι-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, enabling direct comparisons within each sample. Therefore, three different polysaccharide concentration-ionic strength conditions were chosen for each sample, to allow for this comparison. Since sample M’s rheology has been extensively studied under similar conditions, as a carrageenan derived from Mastocarpus (Azevedo et al., 2014, 2022; Moreira et al., 2016; Torres, Chenlo, et al., 2016), 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 (Bui et al., 2019; Ridout et al., 1996). Thermal-rheological Properties of Samples in KCl and NaCl Solutions Each sample was subjected to a time sweep as hot solutions cooled from 85 °C to 25 °C to monitor the sol-gel transition and assess gelation behavior. This approach allows a detailed examination of the evolving viscoelastic properties (G' and G'') during cooling, highlighting gelation temperature (Tg) as the point where G' (storage modulus) crosses G'' (loss modulus), marking the shift from a viscous to a gellike state. The temperature-dependence graphs of the viscoelastic moduli (G' and G'') for selected samples are presented in Figure 13, whereas the cooling curves for all other samples in KCl and NaCl are provided in Figures A.2 and A.3 of Appendix A.2. Notably, different behaviors were observed among the samples. For instance, in the 1 wt.% K+I (10κ90ι) sample in 1M KCl (Figure 13A), G’ and G’’ intersected, allowing for the identification of Tg. In contrast, other samples, such as 2 wt.% KI (sample E) in 0.5M KCl (Figure 13B), maintained a gel state (G’ > G’’) throughout the cooling process, suggesting that Tg occurs above 85 °C. In all cases, the start of gelation (Tstart), marked by a steep increase in G’, was recorded. This approach enables comparison between samples, even when there is no clear crossover point. A third behavior, involving a two-step gelation mechanism was also observed in samples, such as the 2 wt.% KI (sample C) in 1M KCl (Figure 13C). The latter is described as a two-step increase in the viscoelastic moduli, corresponding to independent coil-to-helix transitions of the K and I blocks at distinct temperatures (Tstart1 and Tstart2). This type of gelling behavior has been reported for K+I in the presence of various salts and ionic strengths (Brenner et al., 2014; Bui et al., 2019; Du et al., 2016; Geonzon et al., 70 2019; Hu et al., 2016; Parker et al., 1993; Piculell et al., 1992), but also for K-2 hybrid carrageenans, particularly in the presence of Na+ salts (Hilliou et al., 2014; Hilliou & Gonçalves, 2007; Souza et al., 2011; Torres et al., 2017; van de Velde et al., 2005). Some studies also reported that the step-increase temperatures in moduli for K-2 hybrid carrageenans (Souza et al., 2011, 2023), as well as the temperatures for the coil-to-helix transitions of each blocks (Souza et al., 2011; van de Velde et al., 2005) match those of K+I blends with similar molar compositions, as well as isolated K or I solutions at equivalent concentrations, suggesting that conformational transitions and helical aggregation remain unaffected by the presence of the other component in the blend (Bui et al., 2019; Du et al., 2016; Parker et al., 1993; Souza et al., 2023; van de Velde et al., 2005). Figure 13: Temperature-dependent viscoelastic moduli (G' and G'') of selected carrageenan samples illustrating different gelation behaviors: (A) Gelation temperature (Tg) in 1 wt.% K+I (10κ90ι) in 1 M KCl, where G' and G'' intersect; (B) Absence of Tg for 2 wt.% KI (sample E) in 0.5 M KCl, indicating a gel state maintained throughout cooling; (C) Two-step gelling mechanism observed in 2 wt.% KI (sample C) in 1 M KCl, associated with sequential coil-to-helix transitions in K and I blocks. A B C 71 The values of Tg, Tstart1 and Tstart2, along with their associated errors for each sample, are presented in Table 7 for both KCl and NaCl, as well as a characterization of the gelation mechanism (single-step or two-step process). Table 7: Values of Tg, Tstart1 and Tstart2, along with their associated errors for each sample, for both KCl and NaCl, as well as a characterization of the gelation mechanism (single-step or two-step process) *For these samples, the crossover point Tg was not detectable due to the equipment's limited sensitivity at the deformation level used in the measurement. While this small deformation setting was insufficient to identify Tg, it was chosen to prevent sample disruption and avoid interference with the gelation process. As a result, only the initial gelation temperature Tstart is presented for these samples. Experiment Sample Tg (ºC) Tstart1 (ºC) Tstart2 (ºC) Gelling Mechanism Samples in KCl 2 wt% in 0.01 M KCl Sample C 43.6 ± 0.2 51.0 ± 1.0 31.7 ± 0.1 Two-step 10κ90ι 47.6 ± 0.3 61.0 ± 2.0 34.0 ± 0.5 Two-step 1 wt% in 1 M KCl Sample C -* 74 ± 1.0 60.0 ± 2.0 Two-step 10κ90ι 79.4 ± 0.8 81.0 ± 1.0 46.0 ± 3.0 Two-step 2 wt% in 1 M KCl Sample C 85.4 ± 4.3 > 85 74.0 ± 0.5 Two-step 10κ90ι > 85 > 85 - Single-step 2 wt% in 0.01 M KCl Sample E 66.1 ± 0.2 67.0 ± 0.3 54.0 ± 1.0 Two-step 60κ40ι 47.5 ± 0.4 53.0 ± 1.0 39.5 ± 0.5 Two-step 1 wt% in 0.5 M KCl Sample E > 85 > 85 - Single-step 60κ40ι > 85 > 85 - Single-step 2 wt% in 0.5 M KCl Sample E > 85 > 85 - Single-step 60κ40ι > 85 > 85 - Single-step 0.5 wt% in 0.01 M KCl Sample K -* 43.5 ± 1.5 - Single-step 90κ10ι -* 32.0 ± 1.0 - Single-step 0.5 wt% in 0.5 M KCl Sample K > 85 > 85 - Single-step 90κ10ι > 85 > 85 - Single-step 2 wt% in 0.5 M KCl Sample K > 85 > 85 - Single-step 90κ10ι > 85 > 85 - Single-step Samples in NaCl 2 wt% in 0.01 M NaCl Sample C' 30.7 ± 0.5 38.0 ± 0.5 29.3 ± 0.1 Two-step 10κ90ι 52.0 ± 1.5 55.5 ± 1.0 30.0 ± 1.0 Two-step 1 wt% in 1 M NaCl Sample C' 83.9 ± 3.4 > 85 - Single-step 10κ90ι 55.6 ± 0.3 58.3 ± 0.3 - Single-step 2 wt% in 1 M NaCl Sample C' < 25 25.0 ± 1.0 - Single-step 10κ90ι > 85 > 85 62.0 ± 3.0 Two-step 2 wt% in 0.01 M NaCl Sample K' 39.4 ± 0.4 40.5 ± 0.5 - Single-step 90κ10ι -* 44.5 ± 0.5 - Single-step 1 wt% in 0.5 M NaCl Sample K' -* 53.0 ± 1.0 - Single-step 90κ10ι -* 46.0 ± 0.3 - Single-step 2 wt% in 0.5 M NaCl Sample K' -* 50.0 ± 1.0 - Single-step 90κ10ι -* 54.7 ± 0.3 - Single-step 78 predominantly viscoelastic, characterized by G’’>G’ (Figure 15C). These observations align with the temperature sweep analysis previously conducted for the 2 wt.% sample C’ in 1 M NaCl, which revealed that no gel had formed by the end of the test (T=25 ºC). In the presence of NaCl, while increasing polysaccharide concentration led to an increase in elasticity (wider gap between G’ and G’’) for commercial blends, it did not exert significant differences for hybrid carrageenans C’ and K’. On the other hand, except for commercial blend 90κ10ι, greater salt concentration typically resulted in the loss of elasticity and network stability. However, κ-rich samples consistently resulted in more stable and elastic gels in comparison to ι-rich samples. Gel Properties under Large Deformation In rheological studies of gels, several critical parameters provide insight into their mechanical behavior and structural integrity. The gel elasticity in the linear viscoelastic (LVE) range, represented by G0 (elastic modulus), reflects the gel's stiffness and ability to resist deformation under small applied strains, where the structure remains undamaged. The limiting strain (γL) marks the boundary of this linear range, beyond which the gel structure begins to deform irreversibly or transition into a nonlinear viscoelastic response. To calculate γL, the tolerated deviation from the gel elasticity in the LVE range was defined as 5%, i.e. , all the G’ values below 95% (in strain softening behaviors) or above 105% (in strain hardening behaviors) of the plateau value were considered to be outside of the LVE range. Another crucial point is the flow point (γF), defined by the crossover of storage (G’) and loss moduli (G’’). At this point, the gel transitions from a predominantly solid-like behavior (G’>G’’) to a liquid-like behavior (G’’>G’), representing the onset of flow (Mezger, 2006). A strain sweep test illustrating these parameters is shown in Figure 16A. Additionally, under large deformation, gels can exhibit strain hardening or strain softening behavior, depending on the strand-like structure of carrageenan gels, modulated by the bending rigidity of the filaments and the topology of the network (Carrillo et al., 2013; Meng & Terentjev, 2016; Piculell, 2006). Strain hardening (Figure 16B) refers to an increase in stiffness as strain increases, before rupturing, often indicating strong stretching of filaments. Conversely, strain softening (Figure 16A) reflects a decrease in stiffness with increasing strain, typically associated with structural breakdown or rearrangement (Hilliou, 2021; Mezger, 2006). Practically, these behaviors influence the gel's resilience and functionality in realworld applications, as strain-hardening gels present higher rupture resistance under stress, while strainsoftening gels may fail more easily under high strain conditions. A distinctive behavior where samples presented a strain softening followed by a strain hardening response was also observed in some cases 79 (Figure 16C). Together, these parameters, registered in Table 8, provide a comprehensive understanding of the gel's mechanical properties. Hybrid carrageenans and commercial blends under the same ionic environment and polysaccharide concentration, yielded different mechanical properties, such as strength and deformability. In the LVE region, commercial carrageenans generally produced stronger and stiffer gels (higher G0 values). However, sample E, a K-2 hybrid carrageenan, significantly surpassed the elasticity of its commercial counterpart, under high ionic strengths. The same was also seen occasionally in low ionic strengths ( e.g. , sample C vs. 10κ90ι, in KCl, and sample K’ vs. 90κ10ι, in NaCl). Nonetheless, in most cases, greater elasticity came at the cost of increased gel brittleness ( e.g. , sample E and sample 90κ10ι, in KCl, and sample 10κ90ι, in NaCl). In addition, increasing κ-content, especially in KCl, and increasing polysaccharide concentration usually enhanced gel elasticity. Figure 16: Representation of A) strain softening, B) strain hardening, and C) strain softening followed by strain hardening behaviors. The strain sweep example in A represents a gel-like sample, showing the elastic modulus, (G0), the limiting strain (γL) at the limit of the LVE range, and the flow point (γF) where G’=G’’. γL γF G0 LVE range A B C 80 Table 8: Rheological properties of hybrid carrageenans and commercial blends gelled in KCl and NaCl under varying polysaccharide and ionic conditions. The table includes gel elasticity in the LVE range (G0), limiting strain (γL), and flow strain (γF), as well as strain hardening and softening behaviors under large amplitude oscillatory shear (LAOS). Cases where strain softening is followed by strain hardening are indicated as “No then Yes” Experiment Sample G0 (Pa) γL (%) γF (%) Strain Hardening? Samples in KCl 2 wt% in 0.01M KCl Sample C 2180 ± 17 1.40 ± 0.15 377 ± 30 No 10κ90ι 1022 ± 20 0.69 ± 0.04 118 ± 13 No then Yes 1 wt% in 1M KCl Sample C 48.7 ± 0.3 1.50 ± 0.10 331 ± 14 No 10κ90ι 237 ± 2 1.96 ± 0.13 556 ± 34 No then Yes 2 wt% in 1M KCl Sample C 985 ± 6 3.08 ± 0.18 404 ± 30 No then Yes 10κ90ι 11281 ± 238 0.61 ± 0.03 388 ± 30 No 2 wt% in 0.01M KCl Sample E 7831 ± 23 0.93 ± 0.08 37 ± 5 No 60κ40ι 15351 ± 46 2.04 ± 0.14 60 ± 7 No then Yes 1 wt% in 0.5M KCl Sample E 2391 ± 14 0.94 ± 0.06 35 ± 4 No 60κ40ι 157 ± 1 0.95 ± 0.07 104 ± 10 No 2 wt% in 0.5M KCl Sample E 14860 ± 134 0.30 ± 0.02 11 ± 1 No 60κ40ι 357 ± 2 1.58 ± 0.05 92 ± 4 Yes 0.5 wt% in 0.01M KCl Sample K 490 ± 4 1.38 ± 0.11 123 ± 6 Yes 90κ10ι 535 ± 5 0.96 ± 0.04 136 ± 7 Yes 0.5 wt% in 0.5M KCl Sample K 257 ± 2 0.63 ± 0.05 100 ± 9 No 90κ10ι 1650 ± 61 0.35 ± 0.04 25 ± 2 No 2 wt% in 0.5M KCl Sample K 20476 ± 71 0.81 ± 0.06 18 ± 2 No 90κ10ι 81921 ± 4012 0.37 ± 0.04 3.7 ± 0.6 No Samples in NaCl 2 wt% in 0.01M NaCl Sample C’ 738 ± 3 0.81 ± 0.05 181 ± 5 No 10κ90ι 1400 ± 128 * 0.31 ± 0.02 155 ± 13 No then yes 1 wt% in 1M NaCl Sample C’ 100 ± 60 * <100 * 391 ± 47 No 10κ90ι 800 ± 400 * <1 * 25 ± 3 No 2 wt% in 1M NaCl Sample C’ 80 ± 20 * - * 355 ± 30 No 10κ90ι 410 ± 50 * 4.57 ± 0.23 315 ± 38 No then yes 2 wt% in 0.01M NaCl Sample K’ 18226 ± 111 1.17 ± 0.11 36 ± 5 No 90κ10ι 3832 ± 140 * 1.38 ± 0.02 100 ± 5 Yes 1 wt% in 0.5M NaCl Sample K’ 3384 ± 82 0.64 ± 0.02 41 ± 2 Yes 90κ10ι 6797 ± 45 1.21 ± 0.17 23 ± 1 No 2 wt% in 0.5M NaCl Sample K’ 3118 ± 96 0.35 ± 0.04 11 ± 4 Yes 90κ10ι 25535 ± 311 3.50 ± 0.16 49 ± 7 Yes *The strain sweep analysis of these samples in NaCl revealed certain inconsistencies or experimental artifacts, as evidenced by the absence of a well-defined linear viscoelastic (LVE) region (see Figure A.6, in Appendix A.4). This limitation hindered the accurate determination of key parameters such as the linear elastic modulus (G0) and the limiting strain (γL). The lack of a clear LVE region suggests potential deviations in the gel structure or external factors, such as instrument sensitivity, sample heterogeneity, or ionic interactions specific to NaCl systems. In ι-rich samples in KCl, both salt and polysaccharide concentrations significantly influenced the gels' elastic and rheological properties. At low ionic strength (0.01 M KCl), the hybrid carrageenan (sample C) exhibited higher elasticity and greater resistance to deformation than the commercial blend 10κ90ι. 81 Conversely, at high ionic strength (1 M KCl), the commercial blend consistently displayed greater elasticity. Interestingly, in this ionic environment, the commercial blend at 1 wt.% polysaccharide and sample C at 2 wt.% polysaccharide exhibited strain-hardening behaviors, which coincided with the conditions under which each sample demonstrated their highest resistance to deformation. Despite potential experimental artifacts and inconsistencies in ι-rich samples in NaCl, the commercial blends generally formed stronger gels than the hybrid carrageenans across all conditions studied as shown in Figure A.6, Appendix A.4). At low ionic strength (0.01 M NaCl), the commercial blend also withstood greater strain before permanently losing its linear viscoelasticity. However, at higher ionic strengths, deformation resistance was difficult to evaluate due to irregular and experimentally inaccurate strain sweep graphs. Except for the 1 wt.% polysaccharide experiment at 1 M NaCl, commercial blends and hybrid carrageenans exhibited similar yield points under comparable conditions. To clarify whether these irregularities were due to sample-specific factors or equipment limitations, further analysis under optimized and controlled conditions, using different shearing and/or gelling conditions, is required to ensure reliable and reproducible results and check for possible slip or nonlinear stress response. In the case of sample E and commercial blend 60κ40ι, at low ionic strength, the commercial blend exhibited superior gel elasticity and resistance to mechanical stress compared to the hybrid carrageenan. However, at high ionic strength, sample E showed greater elasticity but endured less deformation, losing structural integrity more easily. Notably, while the gel elasticity of hybrid carrageenan E consistently increased with rising salt concentration, the linear elasticity of the commercial blend 60κ40ι decreased under high ionic strength. This observation aligns with findings by Azevedo et al. (2014), who reported that the linear elasticity (G0) of carrageenan blends increases with salt concentration up to critical point, beyond which the elasticity begins to decline, reflecting a weakening of the gel network. In contrast, the elasticity of hybrid carrageenans followed a different trend, increasing steadily with salt concentration until reaching a saturation point. Beyond this, G0 either became independent of further salt addition or the critical salt concentration laid outside the studied range (0.01–1 M Na⁺ and K⁺). In systems with higher κ-contents, both in KCl and NaCl, the commercial blends consistently exhibited greater elasticity than hybrid carrageenans under equivalent conditions. In KCl, this higher elasticity often came at the cost of increased brittleness. In contrast, in NaCl, commercial blends produced gels that were not only stronger but also softer, demonstrating a higher tolerance for deformation before reaching the irreversible damage limit (γL) and the final breakdown point (γF). 82 Notably, higher κ-contents were also correlated with enhanced gel elasticity when comparing certain commercial blends with lower mol.% κ ( e.g ., 2 wt.% sample 60κ40ι in 0.5 M KCl). These findings align with previous studies documenting the increase in the elastic modulus of mixed gels as κ-carrageenan content rises (Brenner et al., 2014; Rochas et al., 1989; van de Velde et al., 2005), which is further amplified by the presence of potassium ions (Chanvrier et al., 2004; Parker et al., 1993). However, while increasing κ-content significantly increased gel brittleness in commercial blends (consistent with Brenner et al. (2014)), this trend was not observed in hybrid carrageenans ( e.g. , comparing experiments 2 wt.% in 0.5 M KCl, for samples E and K, with 60 and 90 mol.% κ, respectively). These observations suggest fundamental structural differences between the two systems. While hybrid carrageenans displayed lower elasticity, they exhibited an intrinsically higher capacity to resist strain. However, further comparative studies are required to confirm this, particularly involving hybrid carrageenans and commercial blends with varying chemical compositions tested under identical polysaccharide and ionic conditions. Interestingly, both hybrid carrageenans and commercial blends rich in κ exhibited a strain-hardening response at low ionic strengths and a strain-softening response at high ionic strengths, highlighting a shared mechanical behavior despite structural differences. In conclusion, in terms of viscoelastic properties hybrid carrageenans and commercial blends may offer specific advantages depending on the application. While stronger and stiffer gels produced by K+I make them better suited for applications requiring robust structural integrity and minimal deformation, the studied K-2 hybrid carrageenan, for example, showcased its potential for applications where gelling ability resistance to large amount of salt is desirable. However, since increased elasticity often came at the expense of reduced resistance to deformation, leading to greater gel brittleness, it is important to align the choice of carrageenan system with the specific functional requirements of the target application. 4.3 Food Application Given that the food industry is one of the largest markets for carrageenan, and considering the sector's emerging trends, the formulation of functional and vegetarian gummies based on hybrid carrageenans was developed and tested. The significance of simulating a real-world application with hybrid carrageenans lies in the fact that their applicability is not solely dependent on their behavior in the presence of varying ionic strengths, salts, and polysaccharide concentrations, but also on their interactions and synergistic effects with other ingredients, particularly other biopolymers such as xanthan gum. 83 This specific food application was chosen because I had the opportunity to collaborate with Professor Germán Valencia from the Federal University of Santa Catarina (Florianópolis, Brazil) on an ongoing project led by him and his team. In particular, this work aligns with one of their project’s goals: studying the application of a natural biohybrid ingredient, based on anthocyanins extracted from jambolan fruit and adsorbed onto bentonite, in carrageenan hydrogels. The formulation in their work, based on a study by (Song et al., 2022),was adapted to meet the specific objectives of my research and the resources available. Additionally, I recognized that this application aligns with current market trends, serving as a vegetarian alternative to animal-based (gelatin) gummies while tapping into the growing nutraceutical segment focused on functional or fortified gummies (Mahat et al., 2020). Given the global confectionery sector's position as one of the fastest-growing industries (Rawat et al., 2024), I personally saw significant potential and interest in exploring this specific application. The primary goal of this section was to comparatively evaluate the performance of alkaline-treated hybrid carrageenans against both commercial carrageenan blends and untreated hybrid carrageenans. On the one hand, comparing alkali-treated hybrid carrageenans ( i.e. , with biological precursors removed) to commercial blends aimed to assess whether, in a practical application, their performance differs significantly or if they could serve as viable substitutes. On the other hand, we looked into whether the presence of biological precursors in the composition of hybrid carrageenans resulted in a significant difference in gummy performance. This consideration holds industrial interest, as using untreated hybrid carrageenans could reduce processing costs and time. While it is known that alkaline treatment significantly enhances the gelation potential of hybrid carrageenans, when evaluated within a formulation containing other ingredients and biopolymers, these differences may or may not be critical to the final product’s quality. The hybrid carrageenan samples for this application were selected based on results from the phase diagram in KCl (Table 5, Section 4.2.1), as rheological tests were not yet completed at that time. Additionally, time and sample quantity constraints further influenced the selection of samples C, F, and J, limiting the assessment of other hybrid carrageenans. Nevertheless, these three samples, with significantly different κ/ι ratios, provide a general idea of the influence of ι-content on gummy performance when incorporated into a formulation with other biopolymers and functional ingredients. Only samples treated with KOH were tested, as this alkaline solution yielded a more comprehensive gelation profile compared to NaOH (Tables 5 and 6, Section 4.2.1). 84 4.3.1 Particle Size Distribution and Zeta Potential of the Biohybrid The particle size distribution (PSD) and zeta potential (ZP) of the biohybrid material (BH) were assessed to evaluate their implications for stability and dispersion quality in the gummy candy matrix. The particle size distribution of the BH dispersed in water is shown in Figure 17. The particle size distribution of the biohybrid material (bentonite with adsorbed anthocyanins) showed a trimodal distribution, reflecting its complex aggregation behavior. The first peak, around 0.5 μm, likely represents small clusters of bentonite particles. The second dominant peak, at 16-17 μm, corresponds to larger aggregates formed as anthocyanins adsorb onto the clay surface, likely promoting further clustering of particles. The third peak at 140 µm suggests the presence of larger, more loosely associated aggregates, potentially formed during the mixing or drying process. This observed trimodal distribution aligns with behaviors reported in the literature, where bentonitebased materials commonly exhibit a broad particle size range due to hydration, swelling, and aggregation processes (Buntin et al., 2022; Magzoub et al., 2020). Powdered bentonite, rich in montmorillonite, shows a finer and more uniform distribution, with a particle size distribution between 0.5 and 200 µm (Andrade et al., 2021; Vryzas et al., 2016). However, when bentonite is dispersed in water, its particles hydrate and swell, causing significant changes in the size distribution, especially since the clay particles can expand several times their dry size (Magzoub et al., 2020). The larger particle sizes in the biohybrid material could be attributed to the aggregation of montmorillonite layers, especially since anthocyanins adsorb onto the surface, which might bridge the Figure 17: Particle size distribution of the biohybrid material dispersed in an aqueous solution. 85 clay particles and cause clustering. This could also be due to the drying method or other preparation steps, which tend to encourage aggregation. The zeta potential (ZP) of any particle is defined as the number of charges it carries, and, along with particle size distribution, its assessment is a very important parameter for inferring the stability of colloidal dispersions (Parupudi et al., 2022; M. Sharma, 2019; Singhvi et al., 2018). Generally, particles with zeta potential values ranging within − 10 to + 10 mV are considered to have neutral surfaces while those with larger absolute values of 30 mV (+/−) are considered to be strongly cationic or anionic. Therefore, high surface charge on particle surface leads to repulsion and thus prevents aggregation (Mekhamer, 2010; Patravale et al., 2012; Singhvi et al., 2018). The zeta potential of the biohybrid/water system was -19.4 mV, suggesting a moderate level of colloidal stability, with particles likely tending to aggregate over time. This ZP value allows for weak interactions between particles, leading to the formation of larger clusters, particularly in the micron and submicron ranges For a more homogeneous distribution in future applications, improving electrostatic stabilization ( e.g. , by adjusting pH or surface modification (Magzoub et al., 2020; Mekhamer, 2010) could reduce aggregation and result in a more uniform particle size distribution, which is crucial for enhancing the material’s performance in the matrix. 4.3.2 Visual Characterization of Gummy Candy Samples The visual appearance of all GC samples prepared was registered in Figures 18 A-C, for all systems in study, using samples C, F and J, respectively. The carrageenan matrices used enabled gel formation for all GC samples except UT-F (Figure 18B). This outcome is consistent with the balanced composition in ι and κ of the KI, together with the higher content of precursor units (see Figure B.1 in Annex B.1), negatively influencing the gelling behavior. However, it could also be attributed to less careful handling during the physical phase assessment or slight temperature variations, as its counterpart (UT-F + BH) gelled without issue. Alternatively, BH could shift the gel formation conditions as less water is available. In any case, the rheological assessment, conducted under more controlled cooling conditions, will provide further insights into the true gelling behavior of this UT-F sample. 86 Examining the images of the gummy candies, a noticeable color variation can be observed. Specifically, samples without the biohybrid material (BH) exhibited a more yellowish hue, while its addition resulted in a red/pink tone in the untreated KI and commercial K+I samples, and a green tone in the alkaline-treated KI samples. This color variation is related to the BH’s response to different pH environments, due to the incorporation of pH-sensitive anthocyanins (ACNs) (Koop et al., 2024; Merz et al., 2020). The detailed data on the pH values of each gummy candy sample, as well as those of the rosemary honey and biohybrid material used, are gathered in Table A.1, in Appendix A.5. In the presence of the BH, untreated KI and commercial K+I samples showed pH values between 3.6 and 3.9, at which the anthocyanins exhibit a pink hue. In contrast, alkali-treated samples presented higher pH values (around 7.8), where ACNs are oxidized, leading to a blue coloration (Koop et al., 2024). When 10κ90ι 10κ90ι + BH T-C T-C + BH UT-C + BH UT-C T-F UT-F 50κ50ι UT-F + BH T-F + BH 50κ50ι + BH UT-J T-J 90κ10ι UT-J + BH T-J + BH 90κ10ι + BH A B C Figure 18: Images of vegan gummy candies prepared with untreated KI samples (UT-), treated KI samples (T-), and the respective commercial carrageenan blends (mol.% κ; mol.% ι), both with and without the biohybrid material (BH). A) Sample C and Blend 10κ90ι, rich in ι-carrageenan; B) Sample F and Blend 50κ50ι, with a balanced κ/ι ratio; and C) Sample J and Blend 90κ10ι, rich in κ-carrageenan. 87 combined with the yellow matrix of the gummy candies, this blue coloration gave rise to a green color in the alkaline-treated KI samples. The increased pH in the alkali-treated KI samples can be attributed to residual KOH from the treatment process, leaving behind hydroxide ions ([OH⁻]), which raise the pH of the medium, creating a more basic environment. Microscopic images were captured for all GC samples to assess the dispersion quality of the biohybrid material (BH). Since the dispersion behavior was consistent across the three systems (C, F, and J), a detailed discussion will focus on the microscopic images of sample C (Figure 19). These images illustrate differences in the distribution and presence of BH within the matrices. In Figures 19 D, E and F the biohybrid material, colored red or green, appears as distinct aggregates within the matrix, indicating a non-homogeneous dispersion. These aggregates vary in size, with larger clumps and smaller particles dispersed throughout the matrix, in line with the previously discussed particle size distribution of the biohybrid and its zeta potential of -19.4 mV, which reflects moderate colloidal stability. The dispersion, though evident, is incomplete, with particles tending to cluster rather than distribute evenly. With BH Without BH UT-C T-C 10κ90ι A D B C E F Figure 19: Microscopic images of gummy candy samples using untreated carrageenan C (UT-C) (A, D), alkalitreated carrageenan C (T-C) ( B, E), and commercial blend 10κ90ι (C, F), without (A-C) and with (D-F ) the addition of a biohybrid material (BH). The biohybrid, composed of anthocyanins adsorbed onto bentonite, appears as red or green particles (D-F). 94 broader range of strains as κ-content increased, whereas untreated samples tended to lose structural integrity more quickly. Interestingly, the BH reversed these trends: it reduced γL in treated samples but increased it in untreated samples as κ-content rose. These observations highlight the nuanced interplay between chemical composition, BH presence, and gel network behavior. An intriguing strain-hardening response to increasing strain was observed in some samples, particularly those from Group 1 (high ι-content), where samples consistently exhibited softening beyond their LVE region, followed by a transient phase of strain hardening, forming an apparent plateau. This response likely reflects the initial disruption of weaker physical interactions within the gel network, followed by temporary reorganization or alignment of polysaccharide chains into a more stress-resistant configuration, until the eventual collapse of the structure’s integrity. These mechanisms highlight the complexity of carrageenan gel networks, where nonlinear elastic effects and microstructural rearrangements can momentarily counteract deformation before the network ultimately breaks down (Hilliou, 2021; Moraes & Hilliou, 2024). Impact of Carrageenan Source The source of polysaccharide—whether a commercial blend, untreated hybrid carrageenan, or alkalitreated hybrid carrageenan—exerts a notable influence on the rheological properties of GC samples, particularly their elastic modulus and resistance to deformation. Commercial blends consistently exhibited higher G0 values compared to hybrid carrageenan samples, highlighting their ability to form stiffer gels. Among the hybrid samples, alkali-treated carrageenans generally promoted stronger gels relative to untreated samples. However, exceptions to this trend were observed, particularly in Group 1 (high ι-content). In this group, untreated hybrid carrageenan samples ( e.g., UT-C, with or without the BH) surpassed the elastic modulus of their treated counterparts (e.g., TC). Conversely, in Group 2 (balanced κ/ι ratios), the addition of the BH introduced additional complexity, as it altered the expected trends in G0. The variability in the resistance to deformation (γL) further complicates conclusions regarding the impact of the polysaccharide source. The strain resistance of gels appeared to be highly dependent on the chemical composition, including κ/ι ratios, as well as the incorporation of additives such as the BH. This variability underscores the complexity of these systems and the challenge of generalizing the effect of polysaccharide sources on strain resistance. 95 Overall, the choice between untreated and treated hybrid carrageenans should be guided by the specific rheological properties desired for the final formulation. For example, in formulations with higher ι-content, it may not be necessary to use treated hybrid carrageenans to achieve higher G0 values, as untreated samples can sometimes yield comparable or even superior stiffness. However, the decision must also consider the desired strain resistance and other viscoelastic properties, as these are equally influenced by the chemical composition, the presence of biosynthetic precursors, and the inclusion of additives. General Conclusion The interplay between polysaccharide source, chemical composition, and additives must be carefully considered to tailor gel properties to specific applications. Whether the goal is to maximize stiffness, enhance strain resistance, or achieve a balanced combination, each factor must align with the intended functionality of the final product. For gummy formulations, decisions regarding polysaccharide source and treatment are highly context-dependent, requiring a clear understanding of desired textural and mechanical properties. However, while rheological measurements provide essential insights into gel behavior under stress, they capture only part of the story. Additional tests, such as texture profile analysis (TPA), shelf-life studies, microstructural analysis ( e.g. , scanning electron microscopy, SEM) and techniques to assess thermal stability ( e.g. , differential scanning calorimetry, DSC), are needed to fully understand the complexity of texture and stability in food applications (Mahat et al., 2020; Peleg, 2019; V. Sharma & Bhardwaj, 2019). Sensory testing with trained panels or consumer surveys could also provide valuable qualitative feedback on the texture, flavor, and overall satisfaction with the product. Integrating rheological data with these broader testing methods would offer a more comprehensive assessment of gummy performance, ensuring the final product meets both technical requirements and consumer expectations. 4.3.4 Chemical Interactions (FTIR-ATR) The Fourier transform-infrared (FTIR) spectrum is a well-known method for detecting similarities and differences in chemical structures. Understanding whether the addition of a biohybrid material causes structural and chemical changes within the matrix is crucial for industrial applications, as it directly impacts the product's physical properties, stability, and functionality. It also ensures consistency in product quality and aids in predicting scalability challenges. Therefore, the GC samples were characterized using FTIR-ATR to infer possible chemical interactions between the ingredients. 96 This analysis was conducted for all the conditions studied: untreated-KI, alkali treated-KI, and respective commercial K+I blend, with and without the addition of the BH, using KI samples C, F and J. Since the general FTIR spectra was similar for the three systems: C, F and J, only sample J is discussed in detail (Figure 20). The spectra for systems using samples C and F are shown in Figures A.10 and A.11, respectively, in Appendix A.9. The absorbance peaks found in the FTIR spectra (Figure 20) were interpreted considering the ingredients of the formulations: water, carrageenans, xanthan gum, honey and the biohybrid material, composed of bentonite and anthocyanins. Firstly, no differences were reported between samples with and without the BH, confirming that its addition did not cause significant chemical alterations within the matrix. This was an expected outcome due to the low concentration of BH used (only 0.5 wt.%), pointing towards a possible limit of sensitivity of FTIR to detect such chemical interactions. The broad band at 3280 cm-1 is attributed to O-H stretching vibrations, which reflects the presence of water (Ilyas et al., 2016; Kędzierska-Matysek et al., 2018) and hydroxyl groups from carbohydrates (honey, xanthan, and carrageenans) (Damto et al., 2023; Kędzierska-Matysek et al., 2018; Said et al., 2021). The smaller peak found between 2880 and 2920 cm-1 arises from C-H stretching vibrations, indicating the presence of aliphatic chains from sugars in honey and polysaccharides like xanthan gum Figure 20: FTIR-ATR spectra of gummy candies prepared, using untreated carrageenan (UT-J), alkali treated carrageenan (T-J) and the chemical equivalent commercial blend (90κ10ι), with and without the biohybrid material (BH), for sample J. 97 and carrageenans (Kędzierska-Matysek et al., 2018; Pereira, Amado, et al., 2009; Rochas et al., 1986; Said et al., 2021). The prominent peak at 1640 cm-1 corresponds to the O-H bending mode of water (Ilyas et al., 2016; Kędzierska-Matysek et al., 2018). The bands registered between 1350 and 1450 cm-1 are characteristic for bending vibration of O-CH and C-C-H in the carbohydrate structure or bending vibration coming from OH in the C-OH grouping, whereas the band at 1250 cm-1 is, in turn, a vibration characteristic for the stretching vibration of the C-H or CO in carbohydrates (Kędzierska-Matysek et al., 2018, Damto et al., 2023). Another possibility is that these FTIR bands, specifically at 1370 cm-1 and in the 1240–1260 cm-1, range are related to the presence of sulfate esters in carrageenans and other sulfated polysaccharides, arising from vibrational modes of the S=O bond (Pereira, Amado, et al., 2009; Rochas et al., 1986). The visible elbow at approximately 1050 cm-1 (next to the characteristic band at 1025 cm-1) may be due to stretching vibration of C-O-C esters (present in honey and anthocyanins, while the band at 1025 cm-1 is associated with C-O stretching vibrations (Kędzierska-Matysek et al., 2018; Merz et al., 2020; Swer et al., 2018). In the region between 950 and 750 cm-1, the peaks are likely due to C-O-S and C-O stretching from the 3,6-anhydrogalactose units and sulfate groups in carrageenans, as well as saccharide ring vibrations and C–H deformation, characteristic to the anomeric region of carbohydrates (Damto et al., 2023; Gómez-Ordóñez & Rupérez, 2011; Kędzierska-Matysek et al., 2018; Pereira, Amado, et al., 2009). Formulations using alkali-treated carrageenans (T-J and T-J + BH) exhibited slight deviations compared to the other two systems under study. Notably, a minor shift was observed in the peak assigned to the C–H stretching vibration associated with the total sugar content, with a more pronounced band appearing around 2880 cm-1. Additionally, a new peak emerged approximately at 1700 cm-1, and instead of a single peak at 1250 cm-1, the alkali-treated samples displayed two distinct peaks (at around 1200 cm-1 and 1300 cm-1). These observations are consistent with the conversion of hydroxyl groups into carbonyl or ester groups, likely induced by the alkaline environment. The appearance of the carbonyl stretching band (≈1700 cm-1) and the splitting of the C–O stretching region (≈1200–1300 cm-1) are characteristic of esterification or structural rearrangements in polysaccharides (Mobaraki & Hemmateenejad, 2011; Smith, 2018). In conclusion, while the incorporation of the biohybrid material imposed some changes in the rheological behavior of GC formulations, there were no visible differences in the FTIR spectra, indicating that no new chemical bonds were formed. 98 5. CONCLUSIONS AND FUTURE WORK Selected hybrid carrageenans (KI), along with their chemically equivalent commercial blends (K+I), were evaluated through phase diagrams under comparable salt and polysaccharide concentrations. Gel formation occurred more readily in K+I than in KI carrageenans, across almost all chemical compositions (in terms of κ and ι content), polysaccharide concentrations, and ionic strengths, whereas KI carrageenans showed a higher dependency with those parameters. KI carrageenans demonstrated gelation behavior comparable to K+I under specific conditions, such as higher polysaccharide concentrations for ι-rich samples and κ/ι-balanced samples in KCl and NaCl, and lower ionic strengths for κ-rich samples in KCl. K-2 carrageenans displayed salt-specificity, forming gels in KCl but not in NaCl, highlighting their limited gelling potential in Na+ environments. KI and K+I exhibited comparable cooling rheological profiles under identical conditions, particularly in KCl at large ionic strengths. Higher ι-carrageenan content and lower ionic strengths favored two-step gelation mechanisms, while higher κ-content and ionic strengths resulted in single-step gelation. Experimental limitations, such as restricted temperature ranges and strain sensitivity, likely obscured subtle transitions in some systems. Future research should employ advanced rheological techniques with improved control of gelation kinetics for diverse applications. Under Large Amplitude Oscillatory Shear (LAOS) testing KI and K+I yielded distinct mechanical properties under identical ionic environments and polysaccharide concentrations. K+I generally produced stronger and stiffer gels than KI. However, under high ionic strengths, the K-2 hybrid carrageenan significantly outperformed its commercial counterpart in gel elasticity. Similarly, at low ionic strengths, certain hybrids exhibited comparable or even superior elasticity. Increased elasticity often came at the expense of reduced resistance to deformation, leading to greater gel brittleness. This trade-off underscores the importance of aligning the choice of carrageenan system with the specific functional requirements of the target application. Ultimately, the viability of hybrid carrageenans as substitutes for commercial blends depends on the desired balance between gel strength, elasticity, and deformation resistance. In gummy candy formulations, both KI and K+I carrageenans successfully produced gel-like textures across all systems. KI demonstrated potential as effective replacements for K+I, regarding thermal performance, offering a Tg range (65 °C–100 °C) that balances processing flexibility and product stability. In terms of mechanical properties, K+ I produced stiffer gels (G0) with superior strength and elasticity than 99 KI, consistent with prior findings. Alkaline treatment generally enhanced the stiffness and strain resistance of hybrid carrageenans, except for high ι-content samples, where untreated carrageenans outperformed their treated counterparts. This suggests that while precursor removal is often beneficial, the additional alkali treatment step may be unnecessary for specific applications. The incorporation of the biohybrid material often increased stiffness but reduced strain resistance, particularly in treated hybrids and commercial samples with greater κ-content. These findings underscore the intricate interplay between carrageenan source, chemical composition, and additives in shaping gel performance, emphasizing the need for formulation-specific optimization. Hybrid carrageenans show significant potential as clean-label, vegan, and functional food ingredients, combining structural and health-promoting benefits. Refining formulations and processing conditions, alongside further evaluations of texture, thermal stability, and sensory properties, will be key to ensure the final product meets both technical and consumer requirements. 100 BIBLIOGRAPHY Ahmed, A. B. A., Adel, M., Karimi, P., & Peidayesh, M. (2014). Pharmaceutical, Cosmeceutical, and Traditional Applications of Marine Carbohydrates. In Advances in Food and Nutrition Research (Vol. 73, pp. 197–220). Academic Press. https://doi.org/10.1016/B978-0-12-800268-1.00010-X Albuquerque, B. R., Oliveira, M. B. P. P., Barros, L., & Ferreira, I. C. F. R. (2021). Could fruits be a reliable source of food colorants? Pros and cons of these natural additives. Critical Reviews in Food Science and Nutrition , 61 (5), 805–835. https://doi.org/10.1080/10408398.2020.1746904 Alshahrani, A., Alharbi, A., Alnasser, S., Almihdar, M., Alsuhybani, M., & AlOtaibi, B. (2021). Enhanced heavy metals removal by a novel carbon nanotubes buckypaper membrane containing a mixture of two biopolymers: Chitosan and i-carrageenan. Separation and Purification Technology , 276 , 119300. https://doi.org/10.1016/J.SEPPUR.2021.119300 Álvarez-Viñas, M., González-Ballesteros, N., Torres, M. D., López-Hortas, L., Vanini, C., Domingo, G., Rodríguez-Argüelles, M. C., & Domínguez, H. (2022). Efficient extraction of carrageenans from Chondrus crispus for the green synthesis of gold nanoparticles and formulation of printable hydrogels. International Journal of Biological Macromolecules , 206 , 553–566. https://doi.org/10.1016/J.IJBIOMAC.2022.02.145 Álvarez-Viñas, M., Rivas, S., Torres, M. D., & Domínguez, H. (2023). Microwave-Assisted Extraction of Carrageenan from Sarcopeltis skottsbergii. Marine Drugs , 21 (2). https://doi.org/10.3390/md21020083 Álvarez-Viñas, M., Zamboni, F., Domingo, G., Vannini, C., Torres, M. D., Collins, M. N., & Domínguez, H. (2024). Hydrothermal extraction and thorough characterization of carrageenans and proteins from Gigartina pistillata. Food Hydrocolloids , 157 , 110390. https://doi.org/10.1016/J.FOODHYD.2024.110390 Andrade, C., Martínez-Serrano, A., Sanjuán, M. Á., & Tenorio Ríos, J. A. (2021). Reduced Carbonation, Sulfate and Chloride Ingress Due to the Substitution of Cement by 10% Non-Precalcined Bentonite. Materials , 14 (5), 1300. https://doi.org/10.3390/ma14051300 Arias, F., Mansilla, A., Matsuhiro, B., Pavez, J., Torres, R., & Yáñez-Sánchez, M. (2016). Carrageenans from nuclear phases of subantartic Mazzaella laminarioides (Gigartinales, Rhodophyta) and graft copolymerization of alkali-modified carrageenan with acrylamide. Journal of Applied Phycology , 28 (2), 1275–1286. https://doi.org/10.1007/s10811-015-0641-2 101 Avallone, P. R., Russo Spena, S., Acierno, S., Esposito, M. G., Sarrica, A., Delmonte, M., Pasquino, R., & Grizzuti, N. (2023). Thermorheological Behavior of κ-Carrageenan Hydrogels Modified with Xanthan Gum. Fluids , 8 (4), 119. https://doi.org/10.3390/fluids8040119 Azevedo, G., Bernardo, G., & Hilliou, L. (2014). NaCl and KCl phase diagrams of kappa/iota-hybrid carrageenans extracted from Mastocarpus stellatus. Food Hydrocolloids , 37 , 116–123. https://doi.org/10.1016/J.FOODHYD.2013.10.029 Azevedo, G., Domingues, B., Abreu, H., Sousa-Pinto, I., Feio, G., & Hilliou, L. (2015). Impact of cultivation of Mastocarpus stellatus in IMTA on the seaweeds chemistry and hybrid carrageenan properties. Carbohydrate Polymers , 116 , 140–148. https://doi.org/10.1016/J.CARBPOL.2014.04.069 Azevedo, G., Hilliou, L., Bernardo, G., Sousa-Pinto, I., Adams, R. W., Nilsson, M., & Villanueva, R. D. (2013). Tailoring kappa/iota-hybrid carrageenan from Mastocarpus stellatus with desired gel quality through pre-extraction alkali treatment. Food Hydrocolloids , 31 (1), 94–102. https://doi.org/10.1016/J.FOODHYD.2012.10.010 Azevedo, G., Torres, M. D., Almeida, P. L., & Hilliou, L. (2022). Exploring relationships between seaweeds carrageenan contents and extracted hybrid carrageenan properties in wild and cultivated Mastocarpus stellatus, Chondrus crispus and Ahnfeltiopsis devoniensis. Algal Research , 67 , 102840. https://doi.org/10.1016/J.ALGAL.2022.102840 Azevedo, G., Torres, M. D., Sousa-Pinto, I., & Hilliou, L. (2015). Effect of pre-extraction alkali treatment on the chemical structure and gelling properties of extracted hybrid carrageenan from Chondrus crispus and Ahnfeltiopsis devoniensis. Food Hydrocolloids , 50 , 150–158. https://doi.org/10.1016/J.FOODHYD.2015.03.029 Bahari, A., Moelants, K., Huc-Mathis, D., Wallecan, J., Mangiante, G., Mazoyer, J., Hendrickx, M., & Grauwet, T. (2022). Compositional and rheological analysis of carrageenan from the gametophyte phase of the red seaweed Chondrus crispus neutrally extracted at varying temperatures and time. Food Hydrocolloids , 133 , 107995. https://doi.org/10.1016/j.foodhyd.2022.107995 Bahari, A., Moelants, K., Kloeck, M., Wallecan, J., Mangiante, G., Mazoyer, J., Hendrickx, M., & Grauwet, T. (2021). Mechanical disintegration and particle size sieving of Chondrus crispus (Irish moss) gametophytes and their effect on carrageenan and phycoerythrin extraction. Foods , 10 (12). https://doi.org/10.3390/foods10122928 Balasubramanian, R., Kim, S. S., & Lee, J. (2018). Novel synergistic transparent k-Carrageenan/Xanthan gum/Gellan gum hydrogel film: Mechanical, thermal and water barrier properties. International 102 Journal of Biological Macromolecules , 118 , 561–568. https://doi.org/10.1016/J.IJBIOMAC.2018.06.110 Barahona, T., Rodríguez Sánchez, R. A., Noseda, M. D., Mansilla, A., Matsuhiro, B., Prado, H. J., & Matulewicz, M. C. (2021). Characterization of polysaccharides from cystocarpic and tetrasporic stages of Sub-Antarctic Iridaea cordata. Algal Research , 60 , 102503. https://doi.org/10.1016/J.ALGAL.2021.102503 Beaumont, M., Tran, R., Vera, G., Niedrist, D., Rousset, A., Pierre, R., Shastri, V. P., & Forget, A. (2021). Hydrogel-Forming Algae Polysaccharides: From Seaweed to Biomedical Applications. Biomacromolecules , 22 (3), 1027–1052. https://doi.org/10.1021/acs.biomac.0c01406 BeMiller, J. N. (2019). Carrageenans. Carbohydrate Chemistry for Food Scientists , 279–291. https://doi.org/10.1016/B978-0-12-812069-9.00013-3 Bhattacharyya, T., Palla, C. S., Dethe, D. H., & Joshi, Y. M. (2024). Rheological investigation of the network structure in mixed gels of Kappa and Iota Carrageenan. Food Hydrocolloids , 146 , 109298. https://doi.org/10.1016/j.foodhyd.2023.109298 Bhuyan, P. P., Nayak, R., Patra, S., Abdulabbas, H. S., Jena, M., & Pradhan, B. (2023). Seaweed-Derived Sulfated Polysaccharides; The New Age Chemopreventives: A Comprehensive Review. Cancers , 15 (3), 715. https://doi.org/10.3390/cancers15030715 Bianchi, A., Sanz, V., Domínguez, H., & Torres, M. D. (2022). Valorisation of the industrial hybrid carrageenan extraction wastes using eco-friendly treatments. Food Hydrocolloids , 122 , 107070. https://doi.org/10.1016/J.FOODHYD.2021.107070 Bixler, H. J. (2017). The carrageenan controversy. Journal of Applied Phycology , 29 (5), 2201–2207. https://doi.org/10.1007/s10811-017-1132-4 Bixler, H. J., Johndro, K., & Falshaw, R. (2001). Kappa-2 carrageenan: structure and performance of commercial extracts: II. Performance in two simulated dairy applications. Food Hydrocolloids , 15 (4– 6), 619–630. https://doi.org/10.1016/S0268-005X(01)00047-9 Bixler, H. J., & Porse, H. (2011). A decade of change in the seaweed hydrocolloids industry. Journal of Applied Phycology , 23 (3), 321–335. https://doi.org/10.1007/s10811-010-9529-3 Blakemore, W. R. (2016). Polysaccharide Ingredients: Carrageenan. Reference Module in Food Science . https://doi.org/10.1016/B978-0-08-100596-5.03251-0 Boulho, R., Marty, C., Freile-Pelegrín, Y., Robledo, D., Bourgougnon, N., & Bedoux, G. (2017). Antiherpetic (HSV-1) activity of carrageenans from the red seaweed Solieria chordalis (Rhodophyta, Gigartinales) 103 extracted by microwave-assisted extraction (MAE). Journal of Applied Phycology , 29 (5), 2219–2228. https://doi.org/10.1007/s10811-017-1192-5 Brenner, T., Tuvikene, R., Fang, Y., Matsukawa, S., & Nishinari, K. (2015). Rheology of highly elastic iotacarrageenan/kappa-carrageenan/xanthan/konjac glucomannan gels. Food Hydrocolloids , 44 , 136–144. https://doi.org/10.1016/J.FOODHYD.2014.09.016 Brenner, T., Tuvikene, R., Parker, A., Matsukawa, S., & Nishinari, K. (2014). Rheology and structure of mixed kappa-carrageenan/iota-carrageenan gels. Food Hydrocolloids , 39 , 272–279. https://doi.org/10.1016/j.foodhyd.2014.01.024 Bui, V. T. N. T., Nguyen, B. T., Renou, F., & Nicolai, T. (2019). Rheology and microstructure of mixtures of iota and kappa-carrageenan. Food Hydrocolloids , 89 , 180–187. https://doi.org/10.1016/j.foodhyd.2018.10.034 Buntin, A. E., Sirotkin, O. S., & Sirotkin, R. O. (2022). Features of the chemical composition and structure of bentonites in Tatarstan. IOP Conference Series: Earth and Environmental Science , 990 (1), 012041. https://doi.org/10.1088/1755-1315/990/1/012041 Calvo, G. H., Cosenza, V. A., Sáenz, D. A., Navarro, D. A., Stortz, C. A., Céspedes, M. A., Mamone, L. A., Casas, A. G., & Di Venosa, G. M. (2019). Disaccharides obtained from carrageenans as potential antitumor agents. Scientific Reports , 9 (1). https://doi.org/10.1038/s41598-019-43238-y Campo, V. L., Kawano, D. F., Silva, D. B. da, & Carvalho, I. (2009). Carrageenans: Biological properties, chemical modifications and structural analysis – A review. Carbohydrate Polymers , 77 (2), 167– 180. https://doi.org/10.1016/J.CARBPOL.2009.01.020 Cano-Sarmiento, C., Alamilla-Beltrán, L., Azuara-Nieto, E., Hernández-Sánchez, H., Téllez-Medina, D. I., Jiménez-Martínez, C., & Gutiérrez-López, G. F. (2015). High Shear Methods to Produce Nano-sized Food Related to Dispersed Systems (pp. 145–161). https://doi.org/10.1007/978-3-319-135960_8 Cappa, C., Lavelli, V., & Mariotti, M. (2015). Fruit candies enriched with grape skin powders: physicochemical properties. LWT - Food Science and Technology , 62 (1), 569–575. https://doi.org/10.1016/j.lwt.2014.07.039 Carrillo, J.-M. Y., MacKintosh, F. C., & Dobrynin, A. V. (2013). Nonlinear Elasticity: From Single Chain to Networks and Gels. Macromolecules , 46 (9), 3679–3692. https://doi.org/10.1021/ma400478f Castro, A. G. de. (2004). A Reologia. In Instituto Piaget (Ed.), A Química e a Reologia no Processamento dos Alimentos (1st ed., pp. 37–61). Instituto Piaget. 110 for analysis of the quality of honey. BIO Web of Conferences , 10 , 02008. https://doi.org/10.1051/bioconf/20181002008 Khan, S. U., Anjum, S. I., Rahman, K., Ansari, M. J., Khan, W. U., Kamal, S., Khattak, B., Muhammad, A., & Khan, H. U. (2018). Honey: Single food stuff comprises many drugs. Saudi Journal of Biological Sciences , 25 (2), 320–325. https://doi.org/10.1016/j.sjbs.2017.08.004 Khawaja, A. H., Qassim, S., Hassan, N. A., & Arafa, E.-S. A. (2019). Added sugar: Nutritional knowledge and consumption pattern of a principal driver of obesity and diabetes among undergraduates in UAE. Diabetes & Metabolic Syndrome: Clinical Research & Reviews , 13 (4), 2579–2584. https://doi.org/10.1016/j.dsx.2019.06.031 Kim, J. H., Lee, J.-E., Kim, K. H., & Kang, N. J. (2018). Beneficial Effects of Marine Algae-Derived Carbohydrates for Skin Health. Marine Drugs , 16 (11), 459. https://doi.org/10.3390/md16110459 Knutsen, S. H., & Grasdalen, H. (1987). Characterization of Water-extractable Polysaccharides from Norwegian Furcellaria lumbricalis (Huds.) Lamour. (Gigartinales, Rhodophyceae) by IR and NMR Spectroscopy. Botanica Marina , 30 (6), 497–506. https://doi.org/10.1515/botm.1987.30.6.497 Koop, B. L., Soares, L. S., Cesca, K., Souza, V. G. L., Valencia, G. A., & Monteiro, A. R. (2024). Enhancing the stability of anthocyanins extracts through adsorption into nanoclays – development of a smart biohybrid sensor for intelligent food packaging or as natural food additive/preservative. Food and Bioproducts Processing . https://doi.org/10.1016/j.fbp.2024.07.001 Kravchenko, A., Anastyuk, S., Glazunov, V., Sokolova, E., Isakov, V., & Yermak, I. (2023). Structural peculiarities of carrageenans from Far Eastern red seaweed Mazzaella parksii (Gigartinaceae). International Journal of Biological Macromolecules , 228 , 346–357. https://doi.org/10.1016/J.IJBIOMAC.2022.12.167 Kravchenko, A. O., Anastyuk, S. D., Glazunov, V. P., Sokolova, E. V., Isakov, V. V., & Yermak, I. M. (2020). Structural characteristics of carrageenans of red alga Mastocarpus pacificus from sea of Japan. Carbohydrate Polymers , 229 , 115518. https://doi.org/10.1016/J.CARBPOL.2019.115518 Kumari, A., Garima, & Bharadvaja, N. (2023). A comprehensive review on algal nutraceuticals as prospective therapeutic agent for different diseases. 3 Biotech , 13 (2), 44. https://doi.org/10.1007/s13205-022-03454-2 Li, M., Shang, Q., Li, G., Wang, X., & Yu, G. (2017). Degradation of Marine Algae-Derived Carbohydrates by Bacteroidetes Isolated from Human Gut Microbiota. Marine Drugs , 15 (4), 92. https://doi.org/10.3390/md15040092 111 Liu, J., Kandasamy, S., Zhang, J., Kirby, C. W., Karakach, T., Hafting, J., Critchley, A. T., Evans, F., & Prithiviraj, B. (2015). Prebiotic effects of diet supplemented with the cultivated red seaweed Chondrus crispus or with fructo-oligo-saccharide on host immunity, colonic microbiota and gut microbial metabolites. BMC Complementary and Alternative Medicine , 15 (1), 279. https://doi.org/10.1186/s12906-015-0802-5 Lourenço, S. C., Moldão-Martins, M., & Alves, V. D. (2019). Antioxidants of Natural Plant Origins: From Sources to Food Industry Applications. Molecules , 24 (22), 4132. https://doi.org/10.3390/molecules24224132 Magzoub, M. I., Hussein, I. A., Nasser, M. S., Mahmoud, M., Sultan, A. S., & Benamor, A. (2020a). An Investigation of the Swelling Kinetics of Bentonite Systems Using Particle Size Analysis. Journal of Dispersion Science and Technology , 41 (6), 817–827. https://doi.org/10.1080/01932691.2019.1612758 Magzoub, M. I., Hussein, I. A., Nasser, M. S., Mahmoud, M., Sultan, A. S., & Benamor, A. (2020b). An Investigation of the Swelling Kinetics of Bentonite Systems Using Particle Size Analysis. Journal of Dispersion Science and Technology , 41 (6), 817–827. https://doi.org/10.1080/01932691.2019.1612758 Mahat, M. M., Mohmad Sabere, A. S., Shafiee, S. ’Arifin, Nawawi, M. A., Hamzah, H. H., Jamil, M. A. F. M., Che Roslan, N., Abdul Halim, M. I., & Safian, M. F. (2020). The Sensory Evaluation and Mechanical Properties of Functional Gummy in the Malaysian Market . https://doi.org/10.20944/preprints202010.0213.v1 Mahdavi, S. A., Jafari, S. M., Ghorbani, M., & Assadpoor, E. (2014). Spray-Drying Microencapsulation of Anthocyanins by Natural Biopolymers: A Review. Drying Technology , 32 (5), 509–518. https://doi.org/10.1080/07373937.2013.839562 Martín-del-Campo, A., Fermín-Jiménez, J. A., Fernández-Escamilla, V. V., Escalante-García, Z. Y., MacíasRodríguez, M. E., & Estrada-Girón, Y. (2021). Improved extraction of carrageenan from red seaweed (Chondracantus canaliculatus) using ultrasound-assisted methods and evaluation of the yield, physicochemical properties and functional groups. Food Science and Biotechnology , 30 (7), 901– 910. https://doi.org/10.1007/s10068-021-00935-7 Mathlouthi, M., & Koenig, J. L. (1987). Vibrational Spectra of Carbohydrates. In R. S. Tipson & D. Horton (Eds.), Advances in Carbohydrate Chemistry and Biochemistry (1st ed., Vol. 44, pp. 7–89). Academic Press. https://doi.org/10.1016/S0065-2318(08)60077-3 112 Mehvari, F., Ramezanzade, V., An, J., Kim, J., Dinari, M., & Seung Kim, J. (2024). Biopolymer-based hydrogels for biomedical applications: Bioactivity and wound healing properties. Coordination Chemistry Reviews , 518 , 216093. https://doi.org/10.1016/J.CCR.2024.216093 Mekhamer, W. K. (2010). The colloidal stability of raw bentonite deformed mechanically by ultrasound. Journal of Saudi Chemical Society , 14 (3), 301–306. https://doi.org/10.1016/j.jscs.2010.04.013 Meng, F., & Terentjev, E. M. (2016). Nonlinear elasticity of semiflexible filament networks. Soft Matter , 12 (32), 6749–6756. https://doi.org/10.1039/C6SM01029F Merz, B., Capello, C., Leandro, G. C., Moritz, D. E., Monteiro, A. R., & Valencia, G. A. (2020). A novel colorimetric indicator film based on chitosan, polyvinyl alcohol and anthocyanins from jambolan (Syzygium cumini) fruit for monitoring shrimp freshness. International Journal of Biological Macromolecules , 153 , 625–632. https://doi.org/10.1016/j.ijbiomac.2020.03.048 Mezger, T. G. (2006). The Rheology Handbook: For users of rotational and oscillatory rheometers (2nd ed.). Vincentz Network. Mobaraki, N., & Hemmateenejad, B. (2011). Structural characterization of carbonyl compounds by IR spectroscopy and chemometrics data analysis. Chemometrics and Intelligent Laboratory Systems , 109 (2), 171–177. https://doi.org/10.1016/j.chemolab.2011.08.011 Moraes, I. C. F., & Hilliou, L. (2024). Viscoelastic Reversibility of Carrageenan Hydrogels under Large Amplitude Oscillatory Shear: Hybrid Carrageenans versus Blends. Gels , 10 (8). https://doi.org/10.3390/gels10080524 Moreira, R., Chenlo, F., & Torres, M. D. (2016). Gelling characteristics and rheology of kappa/iota-hybrid carrageenans extracted from Mastocarpus stellatus dried at different temperatures. Journal of Applied Phycology , 28 (6), 3635–3644. https://doi.org/10.1007/s10811-016-0879-3 Moses, J., Anandhakumar, R., & Shanmugam, M. (2015). Effect of alkaline treatment on the sulfate content and quality of semi-refined carrageenan prepared from seaweed Kappaphycus alvarezii Doty (Doty) farmed in Indian waters. African Journal of Biotechnology , 14 (18), 1584–1589. https://doi.org/10.5897/AJB2014.14203 Mutlu, C., Tontul, S. A., & Erbaş, M. (2018). Production of a minimally processed jelly candy for children using honey instead of sugar. LWT , 93 , 499–505. https://doi.org/10.1016/j.lwt.2018.03.064 Necas, J., & Bartosikova, L. (2013). Carrageenan: a review. Veterinarni Medicina , 58 (4), 187–205. https://doi.org/10.17221/6758-VETMED 113 Nile, S. H., Baskar, V., Selvaraj, D., Nile, A., Xiao, J., & Kai, G. (2020). Nanotechnologies in Food Science: Applications, Recent Trends, and Future Perspectives. Nano-Micro Letters , 12 (1), 45. https://doi.org/10.1007/s40820-020-0383-9 O’Sullivan, L., Murphy, B., McLoughlin, P., Duggan, P., Lawlor, P. G., Hughes, H., & Gardiner, G. E. (2010). Prebiotics from Marine Macroalgae for Human and Animal Health Applications. Marine Drugs , 8 (7), 2038–2064. https://doi.org/10.3390/md8072038 Pacheco-Quito, E. M., Ruiz-Caro, R., & Veiga, M. D. (2020). Carrageenan: Drug delivery systems and other biomedical applications. In Marine Drugs (Vol. 18, Issue 11). MDPI. https://doi.org/10.3390/md18110583 Padhi, J. R., Nayak, D., Nanda, A., Rauta, P. R., Ashe, S., & Nayak, B. (2016). Development of highly biocompatible Gelatin & i-Carrageenan based composite hydrogels: In depth physiochemical analysis for biomedical applications. Carbohydrate Polymers , 153 , 292–301. https://doi.org/10.1016/J.CARBPOL.2016.07.098 Palachum, W., Klangbud, W. K., & Chisti, Y. (2023). Novel nutritionally-enriched gummy jelly infused with nipa palm vinegar powder and nipa palm syrup as functional food ingredients. Heliyon , 9 (11), e21873. https://doi.org/10.1016/j.heliyon.2023.e21873 Palanisamy, M., Töpfl, S., Aganovic, K., & Berger, R. G. (2018). Influence of iota carrageenan addition on the properties of soya protein meat analogues. LWT , 87 , 546–552. https://doi.org/10.1016/J.LWT.2017.09.029 Parker, A., Brigand, G., Miniou, C., Trespoey, A., & Vallée, P. (1993). Rheology and fracture of mixed ιand κ-carrageenan gels: Two-step gelation. Carbohydrate Polymers , 20 (4), 253–262. https://doi.org/10.1016/0144-8617(93)90097-N Parupudi, A., Mulagapati, S. H. R., & Subramony, J. A. (2022). Nanoparticle technologies: Recent state of the art and emerging opportunities. Nanoparticle Therapeutics: Production Technologies, Types of Nanoparticles, and Regulatory Aspects , 3–46. https://doi.org/10.1016/B978-0-12-8207574.00009-0 Patravale, V., Dandekar, P., & Jain, R. (2012). Characterization techniques for nanoparticulate carriers. In Nanoparticulate Drug Delivery (pp. 87–121). Elsevier. https://doi.org/10.1533/9781908818195.87 Peleg, M. (2019). The instrumental texture profile analysis revisited. Journal of Texture Studies , 50 (5), 362–368. https://doi.org/10.1111/jtxs.12392 114 Pereira, L., Amado, A. M., Critchley, A. T., van de Velde, F., & Ribeiro-Claro, P. J. A. (2009). Identification of selected seaweed polysaccharides (phycocolloids) by vibrational spectroscopy (FTIR-ATR and FTRaman). Food Hydrocolloids , 23 (7), 1903–1909. https://doi.org/10.1016/j.foodhyd.2008.11.014 Pereira, L., Critchley, A. T., Amado, A. M., & Ribeiro-Claro, P. J. A. (2009). A comparative analysis of phycocolloids produced by underutilized versus industrially utilized carrageenophytes (Gigartinales, Rhodophyta). Journal of Applied Phycology , 21 (5), 599–605. https://doi.org/10.1007/s10811009-9447-4 Pereira, L., Sousa, A., Coelho, H., Amado, A. M., & Ribeiro-Claro, P. J. A. (2003). Use of FTIR, FT-Raman and 13C-NMR spectroscopy for identification of some seaweed phycocolloids. Biomolecular Engineering , 20 (4–6), 223–228. https://doi.org/10.1016/S1389-0344(03)00058-3 Pereira, L., & Van De Velde, F. (2011). Portuguese carrageenophytes: Carrageenan composition and geographic distribution of eight species (Gigartinales, Rhodophyta). Carbohydrate Polymers , 84 (1), 614–623. https://doi.org/10.1016/J.CARBPOL.2010.12.036 Perez Recalde, M., Canelón, D. J., Compagnone, R. S., Matulewicz, M. C., Cerezo, A. S., & Ciancia, M. (2016). Carrageenan and agaran structures from the red seaweed Gymnogongrus tenuis. Carbohydrate Polymers , 136 , 1370–1378. https://doi.org/10.1016/J.CARBPOL.2015.10.007 Periyasamy, C., Anantharaman, P., Balasubramanian, T., & Rao, P. V. S. (2014). Seasonal variation in growth and carrageenan yield in cultivated Kappaphycus alvarezii (Doty) Doty on the coastal waters of Ramanathapuram district, Tamil Nadu. Journal of Applied Phycology , 26 (2), 803–810. https://doi.org/10.1007/s10811-014-0256-z Periyasamy, C., & Subba Rao, P. V. (2017). Growth rate and carrageenan yield of cultivated Kappaphycus alvarezii (Doty) Doty in the coastal waters of Bay of Bengal at Chepala Timmapuram, Andhra Pradesh, east coast of India. Journal of Applied Phycology , 29 (4), 1977–1987. https://doi.org/10.1007/s10811-017-1099-1 Piculell, L. (2006). Gelling Carrageenans. In A. M. Stephen, G. O. Phillips, & P. A. Williams (Eds.), Food Polysaccharides and Their Applications (2nd ed., pp. 239–287). CRC Press. https://doi.org/10.1201/9781420015164.ch8 Piculell, L., Nilsson, S., & Muhrbeck, P. (1992). Effects of small amounts of kappa-carrageenan on the rheology of aqueous iota-carrageenan. Carbohydrate Polymers , 18 (3), 199–208. https://doi.org/10.1016/0144-8617(92)90064-W 115 Ponthier, E., Domínguez, H., & Torres, M. D. (2020). The microwave assisted extraction sway on the features of antioxidant compounds and gelling biopolymers from Mastocarpus stellatus. Algal Research , 51 , 102081. https://doi.org/10.1016/J.ALGAL.2020.102081 Porse, H., & Rudolph, B. (2017). The seaweed hydrocolloid industry: 2016 updates, requirements, and outlook. Journal of Applied Phycology , 29 (5), 2187–2200. https://doi.org/10.1007/s10811-0171144-0 Pradhan, B., Bhuyan, P. P., Patra, S., Nayak, R., Behera, P. K., Behera, C., Behera, A. K., Ki, J. S., & Jena, M. (2022). Beneficial effects of seaweeds and seaweed-derived bioactive compounds: Current evidence and future prospective. Biocatalysis and Agricultural Biotechnology , 39 , 102242. https://doi.org/10.1016/J.BCAB.2021.102242 Pradhan, B., & Ki, J. S. (2023). Biological activity of algal derived carrageenan: A comprehensive review in light of human health and disease. International Journal of Biological Macromolecules , 238 , 124085. https://doi.org/10.1016/J.IJBIOMAC.2023.124085 Prasetyaningrum, A., Praptyana, I. R., Nurfiningsih, & Ratnawati. (2019). Carrageenan: Nutraceutical and Functional Food as Future Food. IOP Conference Series: Earth and Environmental Science , 292 (1), 012068. https://doi.org/10.1088/1755-1315/292/1/012068 Premarathna, A. D., Ahmed, T. A. E., Kulshreshtha, G., Humayun, S., Shormeh Darko, C. N., Rjabovs, V., Hammami, R., Critchley, A. T., Tuvikene, R., & Hincke, M. T. (2024). Polysaccharides from red seaweeds: Effect of extraction methods on physicochemical characteristics and antioxidant activities. Food Hydrocolloids , 147 , 109307. https://doi.org/10.1016/J.FOODHYD.2023.109307 Pulidindi, K., & Ahuja, K. (2023). Carrageenan Market Size . https://www.gminsights.com/industryanalysis/carrageenan-market Qiu, L., Zhang, M., Ghazal, A. F., Chu, Z., & Luo, Z. (2024). Development of 3D printed k-carrageenanbased gummy candies modified by fenugreek gum: Correlating 3D printing performance with solgel transition. International Journal of Biological Macromolecules , 265 , 130865. https://doi.org/10.1016/J.IJBIOMAC.2024.130865 Ranneh, Y., Akim, A. M., Hamid, H. Ab., Khazaai, H., Fadel, A., Zakaria, Z. A., Albujja, M., & Bakar, M. F. A. (2021). Honey and its nutritional and anti-inflammatory value. BMC Complementary Medicine and Therapies , 21 (1), 30. https://doi.org/10.1186/s12906-020-03170-5 Ranneh, Y., Ali, F., Zarei, M., Akim, A. M., Hamid, H. A., & Khazaai, H. (2018). Malaysian stingless bee and Tualang honeys: A comparative characterization of total antioxidant capacity and phenolic profile 116 using liquid chromatography-mass spectrometry. LWT , 89 , 1–9. https://doi.org/10.1016/j.lwt.2017.10.020 Rashmi, P., & Mona, K. (2023). Formulation of vegan nutritional gummy supplements and their texturalorganoleptic analysis. Journal of Applied and Natural Science , 15 (4), 1467–1474. https://doi.org/10.31018/jans.v15i4.4857 Rawat, S., Rai, S., Sangeeta, S., Kumar, A., Ramachandran, P., Sharma, S. K., Dubey, S. K., Prakash, A., & Joshi, R. (2024). Application of Plant-Based Hydrocolloids on the Textural Profile of Vegan Gummies Supplemented with Turmeric and Black Pepper. International Journal of Food Science , 2024 , 1–12. https://doi.org/10.1155/2024/7127635 Ridout, M. J., Garza, S., Brownsey, G. J., & Morris, V. J. (1996). Mixed iota-kappa carrageenan gels. International Journal of Biological Macromolecules , 18 (1–2), 5–8. https://doi.org/10.1016/01418130(95)01037-8 Rinaudo, M., & Rochas, C. (1981). Investigations on Aqueous Solution Properties of κ-Carrageenans. In Solution Properties of Polysaccharides (Vol. 150, pp. 367–378). ACS Symposium Series. https://doi.org/10.1021/bk-1981-0150.ch025 Rincones, R. E., Yu, S., & Pedersén, M. (1993). Effect of dark treatment on the starch degradation and the agar quality of cultivated Gracilariopsis lemaneiformis (Rhodophyta, Gracilariales) from Venezuela. Hydrobiologia , 260–261 (1), 633–640. https://doi.org/10.1007/BF00049082 Rioux, L. E., & Turgeon, S. L. (2015). Seaweed carbohydrates. Seaweed Sustainability: Food and NonFood Applications , 141–192. https://doi.org/10.1016/B978-0-12-418697-2.00007-6 Rippe, J., & Angelopoulos, T. (2016). Relationship between Added Sugars Consumption and Chronic Disease Risk Factors: Current Understanding. Nutrients , 8 (11), 697. https://doi.org/10.3390/nu8110697 Rochas, C., Lahaye, M., & Yaphe, W. (1986). Sulfate Content of Carrageenan and Agar Determined by Infrared Spectroscopy. Botm , 29 (4), 335–340. https://doi.org/10.1515/botm.1986.29.4.335 Rochas, C., Rinaudo, M., & Landry, S. (1989). Relation between the molecular structure and mechanical properties of carrageenan gels. Carbohydrate Polymers , 10 (2), 115–127. https://doi.org/10.1016/0144-8617(89)90061-1 Rochas, C., Rinaudo, M., & Landry, S. (1990). Role of the molecular weight on the mechanical properties of kappa carrageenan gels. Carbohydrate Polymers , 12 (3), 255–266. https://doi.org/10.1016/0144-8617(90)90067-3 117 Rodriguez-Amaya, D. B. (2019). Update on natural food pigments - A mini-review on carotenoids, anthocyanins, and betalains. Food Research International , 124 , 200–205. https://doi.org/10.1016/j.foodres.2018.05.028 Rotem, A., Roth‐Bejerano, N., & Arad, S. (Malis). (1986). EFFECT OF CONTROLLED ENVIRONMENTAL CONDITIONS ON STARCH AND AGAR CONTENTS OF GRACILARIA SP. (RHODOPHYCEAE) 1. Journal of Phycology , 22 (2), 117–121. https://doi.org/10.1111/j.1529-8817.1986.tb04153.x Roudbari, M., Barzegar, M., Sahari, M. A., & Gavlighi, H. A. (2024). Formulation of functional gummy candies containing natural antioxidants and stevia. Heliyon , 10 (11), e31581. https://doi.org/10.1016/j.heliyon.2024.e31581 Rudke, A. R., da Silva, M., Andrade, C. J. de, Vitali, L., & Ferreira, S. R. S. (2022). Green extraction of phenolic compounds and carrageenan from the red alga Kappaphycus alvarezii. Algal Research , 67 , 102866. https://doi.org/10.1016/J.ALGAL.2022.102866 Russo Spena, S., Pasquino, R., & Grizzuti, N. (2024). K-Carrageenan/Locust Bean Gum Gels for Food Applications—A Critical Study on Potential Alternatives to Animal-Based Gelatin. Foods , 13 (16), 2575. https://doi.org/10.3390/foods13162575 Said, M., Haq, B., Al Shehri, D., Rahman, M. M., Muhammed, N. S., & Mahmoud, M. (2021). Modification of Xanthan Gum for a High-Temperature and High-Salinity Reservoir. Polymers , 13 (23), 4212. https://doi.org/10.3390/polym13234212 Sanz, V., Torres, M. D., Domínguez, H., Pinto, I. S., Costa, I., & Guedes, A. C. (2023). Seasonal and spatial compositional variation of the red algae Mastocarpus stellatus from the Northern coast of Portugal. Journal of Applied Phycology , 35 (1), 419–431. https://doi.org/10.1007/s10811-02202863-3 Schefer, L., Usov, I., & Mezzenga, R. (2015). Anomalous Stiffening and Ion-Induced Coil–Helix Transition of Carrageenans under Monovalent Salt Conditions. Biomacromolecules , 16 (3), 985–991. https://doi.org/10.1021/bm501874k Shafie, M. H., Kamal, M. L., Zulkiflee, F. F., Hasan, S., Uyup, N. H., Abdullah, S., Mohamed Hussin, N. A., Tan, Y. C., & Zafarina, Z. (2022). Application of Carrageenan extract from red seaweed (Rhodophyta) in cosmetic products: A review. Journal of the Indian Chemical Society , 99 (9), 100613. https://doi.org/10.1016/J.JICS.2022.100613 Shang, Q., Sun, W., Shan, X., Jiang, H., Cai, C., Hao, J., Li, G., & Yu, G. (2017). Carrageenan-induced colitis is associated with decreased population of anti-inflammatory bacterium, Akkermansia 118 muciniphila, in the gut microbiota of C57BL/6J mice. Toxicology Letters , 279 , 87–95. https://doi.org/10.1016/J.TOXLET.2017.07.904 Sharma, G., Khosla, A., Kumar, A., Kaushal, N., Sharma, S., Naushad, M., Vo, D. V. N., Iqbal, J., & Stadler, F. J. (2022). A comprehensive review on the removal of noxious pollutants using carrageenan based advanced adsorbents. Chemosphere , 289 , 133100. https://doi.org/10.1016/J.CHEMOSPHERE.2021.133100 Sharma, M. (2019). Transdermal and Intravenous Nano Drug Delivery Systems. In Applications of Targeted Nano Drugs and Delivery Systems (pp. 499–550). Elsevier. https://doi.org/10.1016/B978-0-12-814029-1.00018-1 Sharma, V., & Bhardwaj, A. (2019). Scanning electron microscopy (SEM) in food quality evaluation. In Evaluation Technologies for Food Quality (pp. 743–761). Elsevier. https://doi.org/10.1016/B9780-12-814217-2.00029-9 Shipp, J., & Abdel-Aal, E.-S. M. (2010). Food Applications and Physiological Effects of Anthocyanins as Functional Food Ingredients~!2009-10-26~!2010-01-06~!2010-03-09~! The Open Food Science Journal , 4 (1), 7–22. https://doi.org/10.2174/1874256401004010007 Shrivastava, A. (2018). Polymerization. In Introduction to Plastics Engineering (pp. 17–48). Elsevier. https://doi.org/10.1016/B978-0-323-39500-7.00002-2 Singhvi, G., Banerjee, S., & Khosa, A. (2018). Lyotropic liquid crystal nanoparticles. In Organic Materials as Smart Nanocarriers for Drug Delivery (pp. 471–517). Elsevier. https://doi.org/10.1016/B9780-12-813663-8.00011-7 Smidsrød, O., & Grasdalen, H. (1984). Polyelectrolytes from seaweeds. In Eleventh International Seaweed Symposium (pp. 19–28). Springer Netherlands. https://doi.org/10.1007/978-94-009-6560-7_3 Smith, B. C. (2018). The C=O Bond, Part VI: Esters and the Rule of Three. Spectroscopy , 33 (7), 20–23. Sokolova, E. V., Chusovitin, E. A., Barabanova, A. O., Balagan, S. A., Galkin, N. G., & Yermak, I. M. (2013). Atomic force microscopy imaging of carrageenans from red algae of Gigartinaceae and Tichocarpaceae families. Carbohydrate Polymers , 93 (2), 458–465. https://doi.org/10.1016/j.carbpol.2012.12.026 Song, H. I., Han, S., Park, J. S., Kim, Y. J., Jeong, C. B., Yarish, C., & Kim, J. K. (2022). Dark treatment effect on the carrageenan characterization in a red alga, Chondrus crispus. Algal Research , 68 , 102889. https://doi.org/10.1016/J.ALGAL.2022.102889 119 Song, X., Chiou, B. Sen, Xia, Y., Chen, M., Liu, F., & Zhong, F. (2022). The improvement of texture properties and storage stability for kappa carrageenan in developing vegan gummy candies. Journal of the Science of Food and Agriculture , 102 (9), 3693–3702. https://doi.org/10.1002/jsfa.11716 Souza, H. K. S., Hilliou, L., Bastos, M., & Gonçalves, M. P. (2011). Effect of molecular weight and chemical structure on thermal and rheological properties of gelling κ/ι-hybrid carrageenan solutions. Carbohydrate Polymers , 85 (2), 429–438. https://doi.org/10.1016/j.carbpol.2011.03.006 Souza, H. K. S., Kraiem, W., Ben Yahia, A., Aschi, A., & Hilliou, L. (2023). From Seaweeds to Hydrogels: Recent Progress in Kappa-2 Carrageenans. In Materials (Vol. 16, Issue 15). Multidisciplinary Digital Publishing Institute (MDPI). https://doi.org/10.3390/ma16155387 Stancioff, D. J. (1981). Reflections on the Interrelationships between Red Seaweed Source, Chemistry and Uses. In T. Levrig (Ed.), International Seaweed Symposium (Xth), Göteborg, Sweden, 11–15 August 1980 (pp. 113–122). De Gruyter. https://doi.org/10.1515/9783110865271-009 Sun, Y., Cui, X., Duan, M., Ai, C., Song, S., & Chen, X. (2019). In vitro fermentation of κ-carrageenan oligosaccharides by human gut microbiota and its inflammatory effect on HT29 cells. Journal of Functional Foods , 59 , 80–91. https://doi.org/10.1016/j.jff.2019.05.036 Swer, T. L., Mukhim, C., Bashir, K., & Chauhan, K. (2018). Optimization of enzyme aided extraction of anthocyanins from Prunus nepalensis L. LWT , 91 , 382–390. https://doi.org/10.1016/j.lwt.2018.01.043 Taghian Dinani, S., Charles Carrillo, M. F., Boom, R., & van der Goot, A. J. (2023). Quality improvement of plant-based meat alternatives by addition of iota carrageenan to pea protein–wheat gluten blend. European Food Research and Technology , 249 (6), 1637–1654. https://doi.org/10.1007/s00217023-04244-7 Tarahi, M., Tahmouzi, S., Kianiani, M. R., Ezzati, S., Hedayati, S., & Niakousari, M. (2023). Current Innovations in the Development of Functional Gummy Candies. Foods , 13 (1), 76. https://doi.org/10.3390/foods13010076 Tasende, M. G., Cid, M., & Fraga, M. I. (2012). Spatial and temporal variations of Chondrus crispus (Gigartinaceae, Rhodophyta) carrageenan content in natural populations from Galicia (NW Spain). Journal of Applied Phycology , 24 (4), 941–951. https://doi.org/10.1007/s10811-011-9715-y Tasende, M. G., Cid, M., & Fraga, M. I. (2013). Qualitative and quantitative analysis of carrageenan content in gametophytes of Mastocarpus stellatus (Stackhouse) Guiry along Galician coast (NW 126 A.3 Mechanical Spectra of Samples in KCl Figure A.4: 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 KCl solutions. Each row represents a different hybrid carrageenan and blend pair, and each column shows results at different concentrations and ionic strengths. Top row - sample C vs. 10κ90ι 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. 60κ40ι 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. 90κ10ι 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. Sample C vs. Blend 10κ90ι Sample E vs. Blend 60κ40ι Sample K vs. Blend 90κ10ι A B C D E F 127 A.4 Large Amplitude Oscillatory Shear Tests A.4.1 Large Amplitude Oscillatory Shear Tests of Samples in KCl Figure A.5: 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. 10κ90ι 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. 60κ40ι 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. 90κ10ι 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. Sample C vs. Blend 10κ90ι, in KCl Sample E vs. Blend 60κ40ι, in KCl Sample K vs. Blend 90κ10ι, in KCl A B C D E F G H I 128 A.4.2 Large Amplitude Oscillatory Shear Tests of Samples in NaCl Figure A.6: 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. 10κ90ι 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; and bottom row - sample K’ vs. 90κ10ι 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. Sample C’ vs. Blend 10κ90ι, in NaCl Sample K’ vs. Blend 90κ10ι, in NaCl A B C D E F 129 A.5 pH Values of Gummy Candies Sample pH (T = 22 ºC) C Untreated UT-C 4.02 UT-C + BH 3.48 Alkali-Treated T-C 7.99 T-C + BH 7.90 Blend 10κ90ι 4.45 10κ90ι + BH 3.84 F Untreated UT-F 4.25 UT-F + BH 3.65 Alkali-Treated T-F 7.58 T-F + BH 7.79 Blend 50κ50ι 4.38 50κ50ι + BH 3.81 J Untreated UT-J 4.32 UT-J + BH 3.71 Alkali-Treated T-J 7.75 T-J + BH 7.64 Blend 90κ10ι 4.73 90κ10ι + BH 4.01 Rosemary Honey 3.54 BH + Distilled Water 3.88 Table A.1: pH values of gummy candies prepared with and without the biohybrid material (BH), as well as the pH of rosemary honey and the biohybrid used in the preparations 130 A.6 Temperature Dependence of the Storage (G’) and Loss (G’’) Moduli in Gummy Candies Figure A.7: Temperature dependence of the storage modulus (G’, full squares) and loss modulus (G’’, open squares) of gummy candy samples. Results are presented for commercial blends (in red), alkali-treated (T-) hybrid carrageenans (in blue) and untreated (UT-) hybrid carrageenans (in green). Each row represents a different set of samples based on their chemical composition: top row (A, B) 10κ90ι, T-C and UT-C; middle row (C, D) 50κ50ι, T-F and UT-F; bottom row (E, F) 90κ10ι, T-J and UT-J. Graphs in the right column (B, C, D) show the effects of incorporating the biohybrid material (+BH). A ( γ L ) B ( γ L ) C ( γ L ) D ( γ L ) E ( γ L ) F ( γ L ) 131 A.7 Mechanical Spectra of Gummy Candies Figure A.8: Mechanical spectra of gummy candy samples, showing the storage modulus (G’, full squares) and loss modulus (G’’, open squares) as a function of frequency variation. Results are presented for commercial blends (in red), alkali-treated (T-) hybrid carrageenans (in blue) and untreated (UT-) hybrid carrageenans (in green). Each row represents a different set of samples based on their chemical composition: top row (A, B) 10κ90ι, T-C and UT-C; middle row (C, D) 50κ50ι, T-F and UT-F; bottom row (E, F) 90κ10ι, T-J and UT-J. Graphs in the right column (B, C, D) show the effects of incorporating the biohybrid material (+BH). A B C D E F 132 A.8 Large Amplitude Oscillatory Shear Tests of Gummy Candies Figure A.9: Large amplitude oscillatory shear (LAOS) tests showing the storage modulus (G’, full squares) and loss modulus (G’’, open squares) as a function of applied strain for gummy candy samples. Results are presented for commercial blends (in red), alkali-treated (T-) hybrid carrageenans (in blue) and untreated (UT-) hybrid carrageenans (in green). Each row represents a different set of samples based on their chemical composition: top row (A, B) 10κ90ι, T-C and UT-C; middle row (C, D) 50κ50ι, T-F and UT-F; bottom row (E, F) 90κ10ι, T-J and UT-J. Graphs in the right column (B, C, D) show the effects of incorporating the biohybrid material (+BH). A B C D E F 133 A.9 FTIR-ATR Spectra of Gummy Candies A.9.1 FTIR-ATR Spectra of Gummy Candies, for systems using Sample C and Commercial Blend 10κ90ι A.9.2 FTIR-ATR Spectra of Gummy Candies, for systems using Sample F and Commercial Blend 50κ50ι Figure A.10: FTIR-ATR spectra of gummy candies prepared, using untreated carrageenan (UT-C), alkali treated carrageenan (T-C) and the chemical equivalent commercial blend (10κ90ι), with and without the biohybrid material (BH), for sample C. Figure A.11: FTIR-ATR spectra of gummy candies prepared, using untreated carrageenan (UT-F), alkali treated carrageenan (T-F) and the chemical equivalent commercial blend (50κ50ι), with and without the biohybrid material (BH), for sample F. 134 B. ANNEXES B.1 Chemical Composition of Untreated Hybrid Carrageenans B.2 Molecular Mass Distribution of Untreated Hybrid Carrageenans and Commercial Pure Kand I-carrageenans Figure B.1: Chemical composition of untreated hybrid carrageenan samples. Figure B.2: Molecular mass distribution (Mw) and polydispersity index (PDI) of untreated samples (A–L), as well as commercial pure Kand I-carrageenans.