Sequential extraction of natural products from carrageenophytes: focus on hybrid carrageenans and the impact of the molecular mass on their gel properties

University of Minho School of Engineering Maria Gabriela Afonso Gonçalves Sequential extraction of natural products from three carrageenophytes: focus on hybrid carrageenans and the impact of molecular mass on their gel properties December 2024 Sequential extraction of natural products from three carrageenophytes: focus on hybrid carrageenans and the impact of molecular mass on their gel properties Maria Gabriela Afonso Gonçalves UMinho | 2024 Maria Gabriela Afonso Gonçalves Sequential extraction of natural products from carrageenophytes: focus on hybrid carrageenans and the impact of the molecular mass on their gel properties December 2024 Master’s Dissertation Masters in Biological and Chemical Engineering Dissertation supervised by Loïc Hilliou, PhD Bruno Faria, PhD 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 users of this work: Attribution CC BY https://creativecommons.org/licenses/by/4.0/ iii ACKNOWLEDGEMENTS I am deeply grateful to everyone who, in some way, contributed to the realization of this thesis. This work is not only the culmination of six months or five years but a testament of my continuous growth as a professional and as a person. First and foremost, my sincere thanks to Professor Dr. Loic Hilliou from the Institute of Polymers and Composites at the University of Minho, for welcoming me into his research group and for his unwavering belief in my capabilities. I am grateful for his straightforward and scientific approach during our meetings, never allowing discouragement to take hold, even when faced with setbacks. His flexibility in letting me shape the project's focus to align with my interests was truly invaluable. I would also like to express my appreciation to Professor Dr. Bruno Faria, whose insightful conversations pushed me to view my work from fresh perspectives, always driving its improvement. I am immensely thankful to my laboratory and course colleague, Maria Alice Monteiro, for her friendship, generosity and the support she offered with such warmth. Each conversation with her was a source of motivation that recharged me. I also extend my thanks to Rui Ribeiro, Izabel Moraes, João Alves and Margarida Gonçalves, whose assistance was indispensable to the progress of my laboratory experiments. To my dear friends, I am profoundly grateful for your presence and constant encouragement. Thank you for believing in me, even in moments of doubt, and for always being there to lift me up when I felt overwhelmed. Your unwavering support, the moments of laughter, and the shared dreams were what kept me going. I carry a deep appreciation for each of you. Finally, and most importantly, I wish to express my profound gratitude to my parents, my sister and my grandparents. Mom and Dad, thank you for your unconditional love, for always being my safe harbour, and for instilling in me the resilience to chase my dreams without fear. Xana, thank you for being my source of endless inspiration. And grandpas, thank you very much for everything, your faith in me is a gift beyond words. This work was supported by the Fundação para a Ciência e Tecnologia (FCT), through the E2B2PHACAR project (http://doi.org/10.54499/PTDC/BII-BIO/5626/2020). Additional financial support by the FCT under the framework of Strategic Funding grant: UID/CTM/50025/2020 and grant: CEECINST/00156/2018 (https://doi.org/10.54499/CEECINST/00156/2018/CP1642/CT0012) are also acknowledged. To all of you, my deepest thanks. iv 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, December 2024 Maria Gabriela Afonso Gonçalves v RESUMO Extração sequencial de produtos naturais de três carragenófitas: foco em carrageninas híbridas e o impacto da massa molecular nas suas propriedades de gelificação As carrageninas são polissacarídeos sulfatados conhecidas pelas suas propriedades de gelificação termorreversíveis, amplamente utilizadas nas indústrias alimentar, farmacêutica e cosmética. Recentemente, as carrageninas híbridas (K2) têm despertado interesse devido às suas propriedades de gelificação intermediárias. Este estudo tem como objetivo desenvolver um método de extração sequencial (SE) para algas vermelhas ( Chondrus crispus , Mastocarpus stellatus , Gigartina pistillata ), alinhado ao conceito de biorrefinaria, por extrusão extrativa. Os principais objetivos deste trabalho incluem a extração e caracterização de K2-carrageninas, avaliando o impacto de extrações prévias e o tempo de extração. Adicionalmente, pretende-se estudar o efeito da redução de massa molecular (Mw) e determinar uma massa molecular crítica (Mc) para a gelificação da K2-carragenina. Para isolar ficobiliproteínas, clorofilaa, carotenoides e carrageninas das algas, foi utilizada a seguinte sequência: extração com água fria (CWE), extração etanólica (EE), extração com água quente (HWE) e extração alcalina quente (HAE). O rendimento foi determinado para cada fração, e os extratos de carrageninas foram analisados quanto à Mw, aos níveis de sulfatação (FTIR, 1H-NMR) e às propriedades reológicas dos seus géis e líquidos resultantes. A redução da Mw foi alcançada através de ultrassonicação, com os produtos degradados sujeitos a análises semelhantes às anteriores. Os resultados indicam que a SE separa eficazmente os componentes das algas, facilitando a recuperação de carrageninas com alta Mw, particularmente em M. stellatus e G. pistillata , com elasticidade de gel (G’) e viscosidade superiores em comparação com os controlos. A HWE produz carrageninas de alto Mw e com propriedades reológicas melhoradas, enquanto os extratos HAE apresentam menor Mw e potencial de gelificação reduzido. Em geral, extrações prolongadas melhoram a Mw e a G', embora C. crispus tenha mostrado uma tendência distinta. Os resultados da Mw destacam que o ultrassom leva à diminuição e dessulfatação das carrageninas ao longo do tempo de processamento. Ademais, a Mc depende da composição química, sugerindo uma Mc de ~1·10⁵ g/mol para híbridas ricas em κ e ~3·10⁵ g/mol para híbridas ricas em ι, além das quais a degradação adicional tem impacto mínimo na gelificação. Em suma, este estudo demonstra o potencial de adaptação de protocolos de extração para atender às necessidades industriais, contribuindo para a valorização sustentável das algas vermelhas através de métodos de biorrefinaria. Palavras-chaves: Carrageninas híbridas; extração sequencial; massa molecular; reologia. vi ABSTRACT Sequential extraction of natural products from three carrageenophytes: focus on hybrid carrageenans and the impact of molecular mass on their gel properties Carrageenans are sulphated polysaccharides known for their thermo-reversible gelling properties, widely used in the food, pharmaceutical, and cosmetic industries. Hybrid carrageenans (K2) have recently gained interest for their intermediate gelling properties. This study aims to develop a sequential extraction (SE) method for red seaweeds ( Chondrus crispus, Mastocarpus stellatus, Gigartina pistillata ), aligning with the concept of extractive extrusion biorefinery. The primary objectives of this study include extracting and chemically characterizing K2-carrageenans, evaluating the impact of prior extractions and time of extraction. Additionally, this work seeks to assess the influence of molecular mass (Mw) reduction on gelling properties and to determine a critical molecular mass (Mc) for gelation. To isolate phycobiliproteins, chlorophyll-a, carotenoids, and carrageenans from the algae, the following sequence was used: cold-water extraction (CWE), ethanolic extraction (EE), hot water extraction (HWE), and hot alkaline extraction (HAE). Each fraction's yield was determined, with carrageenan extracts analysed for Mw, sulphation levels (trough FTIR, 1H-NMR), and their rheological properties in gels and liquids. Molecular mass reduction was achieved through ultrasonication, and the degraded products were characterized in a similar manner as before. Results indicate that SE effectively separates algal components, facilitating the recovery of high-Mw carrageenans, particularly in M. stellatus and G. pistillata , with superior gel elasticity (G’) and viscosity compared to the controls. HWE yields high-Mw carrageenans with enhanced rheological properties, whereas HAE extracts exhibit lower Mw and reduced gelation potential. Prolonged extraction generally improves Mw and G’, although C. crispus showed a distinct trend. The Mw results highlight that ultrasonication leads to the degradation and desulphation of carrageenans over processing time. Furthermore, the Mc depends on the chemical composition, suggesting an Mc of ~1·10⁵ g/mol for κ-rich hybrids and ~3·10⁵ g /mol for ι-rich hybrids, beyond which further degradation has minimal impact on gelation. In summary, this study demonstrates the potential for adapting extraction protocols to meet industrial demands, contributing to the sustainable valorisation of red algae through biorefinery methods. Keywords: Hybrid carrageenan; molecular mass; rheology; sequential extraction. vii CONTENTS 1. Introduction .............................................................................................................................. 17 1.1 Context .............................................................................................................................. 17 1.1.1 Carrageenans ............................................................................................................. 17 1.1.2 Project E2B2 – PHACAR ............................................................................................. 17 1.2 Motivation .......................................................................................................................... 18 1.2.1 Biorefinery concept ..................................................................................................... 18 1.2.2 Impact of Mw in the rheological properties .................................................................. 19 1.3 Goals ................................................................................................................................. 19 1.4 Structure of the dissertation ............................................................................................... 20 2. State of art ................................................................................................................................ 21 2.1 Red Algae .......................................................................................................................... 21 2.1.1 Composition ............................................................................................................... 22 2.1.2 Pigments of Red Algae ................................................................................................ 24 2.2 Carrageenan ...................................................................................................................... 27 2.2.1 Relevance .................................................................................................................. 27 2.2.2 Chemical structure of carrageenans ............................................................................ 28 2.2.3 Extraction ................................................................................................................... 30 2.2.4 Gel Properties ............................................................................................................ 32 2.2.5 Molecular Mass, Mw ................................................................................................... 34 2.3 Sequential Extraction ......................................................................................................... 37 2.3.1 Biorefinery Strategies for the Valorisation of Red Seaweeds ......................................... 38 2.3.2 Extractive – Extrusion ................................................................................................. 40 3. Materials and Methods .............................................................................................................. 42 xiv ACRONYMS 1H-NMR Proton Nuclear Magnetic Resonance AFM Atomic Force Microscopy ATR Attenuated Transmission Reflectance CAGR Compound Annual Growth Rate Carot Carotenoid Chloro-a Chlorophyll-a Cont Control CWE Cold Water Extraction D-unit α-D-galactopyranose d.w. Dry Weight DA-units 3,6-anhydrogalactose E2B2-PHACAR Extractive Extrusion-Based Biorefinery of polyhydroxyalkanoates and carrageenans EE Ethanolic Extraction FTIR Fourier Transform Infrared Spectroscopy G-unit β-D-galactopyranose G’ Dynamic/elastic moduli G’’ Loss moduli G0 G’ at 1 Hz at 25ºC of gels G1 G’ at 1Hz and 25 ºC of liquids ɣ Shear strain HAE Hot Alkaline Extraction HWE Hot Water Extraction I Iota (a type of carrageenan) K Kappa (a type of carrageenan) K2 Hybrid K/I (a type of carrageenan) xv L Lambda MAE Microwave-Assisted Extraction Mc Critical Molecular Mass Mn Number-average Molecular Mass Mu G4S-D6S Mw Molecular Mass Mwgel Mw required for gel formation Nu G4S-D2S,6S PDI Polydispersity Index PUFAs Omega-3 and Omega-6 polyunsaturated fatty acids R-PE R-phycoerythrin RTD Residence Time Distribution SE Sequential Extraction t Time T Temperature Tg Gelation Temperature Tm Melting Temperature TON Onset Temperature UAE Ultrasound-Assisted Extraction wt.% % Total Weight η Apparent Shear Viscosity ι G4S-DA2S κ G4S-DA ω Frequency xvi LIST DE SYMBOLS h Hours min Minutes ºC Degrees Celsius 17 1. INTRODUCTION 1.1 Context 1.1.1 Carrageenans Carrageenans are sulphated polysaccharides, present in the cell walls of red algae ( Rhodophyta ) (Abdul et al., 2018; Campo et al., 2009; Shukla et al., 2016). They are commercially important due to their thermo-reversible gelling properties (Hilliou, 2021), and widely used as food additives (E407) for thickening, stabilizing, and gelling, in products like ice cream, sauces, meat, and as a vegan gelatine alternative (Souza et al., 2023; Stephen et al., 2006). The unique characteristics of carrageenans arise from variations in their molecular structure, including different sulphate groups and molecular mass. Understanding these properties is essential for tailoring their application in various fields, especially given the increasing demand for natural and sustainable bio-based products. 1.1.2 Project E2B2 – PHACAR This study is part of the E2B2-PHACAR project, which aims to develop a novel biorefinery concept called the Extractive Extrusion-Based Biorefinery (E2B2). The E2B2 approach focuses on the continuous and sequential extraction of a range of valuable products, including polyhydroxyalkanoates (PHA), natural compounds, and gelling hybrid carrageenans (CAR). So far, commercial extruders have been employed for pre-treatment of very few biomasses (Vauchel et al., 2008) or for the extraction of single products (Sugiono et al., 2019). The E2B2 project seeks to expand these applications by feeding biomass into an extruder, to extract multiple products in a cascade manner, as seen in Figure 1. Within this framework, fermentation broths of PHA from mixed microbial culture and seaweeds containing hybrid carrageenans are being studied. The research team has confirmed the block copolymer structure of K2-carrageenans (Souza et al., 2023) and identified complex interactions between their chemical composition and the mechanical and structural properties of the resulting gels (Hilliou, 2021). However, the exact mechanism and structural characteristics of K2-carrageenan gels remain unresolved. Addressing these challenges is the second key objective of the E2B2-PHACAR project. 18 Figure 1. Model of the extractive-extrusion concept of the E2B2-PHACAR project. 1.2 Motivation 1.2.1 Biorefinery concept Seaweeds possess unique bioactive components not found in terrestrial biomass, positioning them as an exceptional and sustainable resource. Their potential is particularly evident in seaweed-based biorefineries, which align with the principles of circular economy and sustainability by promoting the efficient use of resources, reducing environmental impact, and contributing to job creation and economic growth. This is also in line with key Sustainable Development Goals outlined by the United Nations, such as clean energy, economic growth, resource-use efficiency, responsible consumption and production, and climate action. (Álvarez-Viñas et al., 2019; Balina et al., 2017; Torres, Kraan, et al., 2019). Sequential extraction as a biorefinery approach holds promise by allowing for the recovery of multiple valuable products. However, as said before, commercial extruders have traditionally been limited to delivering a single extract and an extrudate. Establishing a continuous biorefinery concept capable of refining biomass into a cascade of products directly along the extruder is a goal that remains unexplored (see Figure 1). In the current state, K2-carrageenans are batch-extracted through a resource-intensive process, involving alkali pre-treatment followed by hot water extraction (Bianchi et al., 2022; Souza et al., 2023). Such methods require substantial chemical inputs and generate significant waste. The E2B2 project addresses these limitations by investigating the feasibility of sequential extraction within an extruder, where seaweed is processed with water and alkali, allowing for multiple products to be recovered with 19 minimal environmental footprint compared to conventional methods. Although E2B2’s application has so far focused on K2-carrageenans, red seaweeds also contain other valuable components, such as proteins and pigments, which could be explored in future developments. 1.2.2 Impact of Mw in the rheological properties The molecular mass (Mw) of carrageenans plays a pivotal role in determining their gelling properties, including elasticity and mechanical strength (Souza et al, 2023). In the context of the E2B2PHACAR project, understanding how Mw variations affect the gels mechanical properties is crucial, as the project aims to refine and expand the sequential extraction process, and the extraction conditions directly influence the Mw of carrageenans. However, despite its importance, the literature lacks comprehensive studies specifically on the Mw-dependent gelling behaviour of K2-carrageenans. As part of the second key objective of the E2B2-PHACAR project, this study aims to address these knowledge gaps by exploring the relationship between Mw and gel formation, focusing on K2-carrageenans extracted through this novel biorefinery concept. 1.3 Goals The primary goal of this thesis is to design a preliminary procedure for the sequential extraction of K2-carrageenans, to be used later with an extruder under the extractive-extrusion technique, in alignment with the objectives of the E2B2-PHACAR project. The aim is to extract various products from algal biomass using different solvents and extraction conditions, and to chemically and rheological characterize the K2carrageenans obtained, assessing the impact of prior extractions on sequential products. Furthermore, this study seeks to establish relationships between extraction duration and gel elasticity. The secondary goal of this thesis is to evaluate the effect of molecular mass reduction on the gelling properties of K2-carrageenans, using ultrasonication. Additionally, it seeks to determine whether a critical molecular mass (Mc) for K2-carrageenan gelation exists and, if so, to identify this threshold. 20 1.4 Structure of the dissertation This document is organized as follows: • 2. State of the Art: This chapter provides a comprehensive overview of the key topics relevant to this dissertation. It begins by discussing the composition of red algae and exploring the different types and chemical structures of carrageenans, with a particular focus on K2-carrageenans. It also covers current extraction methods and the existing knowledge on the influence of molecular mass on gel properties. Additionally, it introduces the concept of sequential extraction and the extractive-extrusion technique, situating these approaches within the broader context of sustainable and efficient biorefinery practices. • 3. Materials and Methods. This chapter details the materials and methodologies employed to carry out the proposed tasks in this study. It includes a thorough description of the sequential extraction procedure, and the characterization techniques used to analyse the extracted and ultrasonicated K2-carrageenans. • 4. Results and Discussion. This section presents the results obtained throughout the study, accompanied by an in-depth analysis and discussion. It begins with the yield of extracted products, followed by the rheological and chemical properties of the various carrageenan extracts. Then, the next part addresses the impact of ultrasonication on K2-carrageenans, examining molecular mass and rheological behaviour to provide a holistic understanding of the effects of molecular breakdown. • 5. Conclusions and future work. This concluding chapter summarizes the key findings of the study, emphasizing the main contributions and insights gained. It also outlines potential avenues for future research, focusing on unresolved challenges and opportunities for advancing the sequential extraction process and optimizing carrageenan gel properties. 21 2. STATE OF ART Marine macroalgae, commonly known as seaweed, are complex photosynthetic organisms present in various aquatic environments. They have been common since ancient times for alimentary and medicinal purposes, especially in Asian countries (Barbot et al., 2016). Advancements in scientific research and novel methodologies have facilitated the identification of compounds derived from seaweed. These compounds have been extensively studied for their potential therapeutic effects on the human body, including their antioxidant, antimicrobial, and antitumoral properties (Lomartire et al., 2021). Moreover, seaweeds might be also employed in several industrial applications, such as the production of fertilizers (Balina et al., 2017; Torres, Kraan, et al., 2019), aquaculture feed, biofuels, in wastewater treatments or to produce innovative, and ecologic materials to replace plastic equivalents (Kim et al., 2017; Raikova et al., 2019), contributing towards a sustainable solution to protect the environment from the discharge of non-biodegradable plastic. Also, marine macroalgae present economic and environmental advantages over terrestrial biomass, including rapid growth rate, no competition for agricultural land and the depolymerisation of seaweeds before the biological conversion into biofuels is facilitated due to the high carbohydrate and low lignin content, among other advantages (Álvarez-Viñas et al., 2019). So, seaweeds are an interesting option for the biorefinery concept. Seaweeds are classified into three main groups according to their colour, which is determined by the presence of certain pigments. Brown seaweeds (phylum Ochrophyta , class Phaeophyceae ) have an abundance of pigments that vary from yellow to dark brown (Seely et al., 1972). Red seaweeds (phylum Rhodophyta ) are characterized by their high amounts of carotenoids, chlorophyll-a, phycoerythrin, phycocyanin, and allophycocyanin (Denis et al., 2009). Green seaweeds (phylum Chlorophyta ) possess mainly the pigment chlorophyll, which has a crucial role in photosynthesis and gives the characteristic green colour to plants, algae, and cyanobacteria (Aryee et al., 2018). 2.1 Red Algae Red seaweeds are of economic importance, they represent 61 % of global seaweed production (Penuela et al., 2018), and are primarily used for extracting agar and carrageenan. Moreover, they also contain other compounds of interest, in amounts varying qualitatively and quantitatively among different 22 species, especially since the extraction method is not generally standardized and for the fact that various abiotic and biotic factors, such as the growth stage, harvest location and period, depth, nutrient quantity and quality, temperature, salinity, light exposure, may also differ (Generalić Mekinić et al., 2023). To provide a comprehensive understanding of the red seaweed, the following section will first explore the general composition of red algae, before focusing on specific compounds relevant to this study (pigments and carrageenans). These compounds will be studied in three distinct red algal species: Chondrus crispus , Gigartina pistillata , and Mastocarpus stellatus. Providing a valuable opportunity to explore their potential within the framework of a biorefinery approach. 2.1.1 Composition It is fundamental to have some knowledge about the composition of the red algae and their most studied biological properties, to pave the way for the utilization of this resource. So, in the next lines the general composition of the red algae is discussed. Carbohydrates According to their prevalence in algal sources, agar and carrageenan, both sulphated polysaccharides, known as phycocolloids, are the most relevant carbohydrates in red seaweeds, accounting for up to 40–50 % of their dry weight (d.w.) (Torres, Flórez-Fernández, et al., 2019) followed by other polysaccharides found in significantly lower amounts, such as xylans, sulphated galactans and porphyrans (Øverland et al., 2019). Proteins Among the different algae classes found in marine ecosystems, red seaweeds exhibit the highest content of proteins (Belghit et al., 2017; Øverland et al., 2019). Generally, the protein content of algae ranges between 5 % and 20 %, although red algae may achieve greater proportions, with maximum values reaching 47 % of total d.w. (Cian et al., 2012; Praveen et al., 2019; Rudtanatip et al., 2018). Nevertheless, proteins depict a species-dependent occurrence, considering that some species, such as those from Gracilaria genus present a low protein content (below 5 % d.w.) whereas others like Pyropia tenera show a protein content of 37 % d.w. (Holdt & Kraan, 2011). Furthermore, in terms of total proteins, phycobiliproteins are the predominant proteins in red seaweeds, constituting up to 50 % of the total 23 protein content and contributing to the reddish coloration characteristic of these algae (Niu et al., 2010). Regarding the amino acid composition of red algae proteins, a high concentration of essential amino acids has been documented, with aspartic acid and glutamic acid being the most abundant residues, comprising 22–44 % of the total amino acids found in red algae proteins (Cian et al., 2012). Lipids Despite low lipid content, red algae are rich in nutritionally important PUFAs (Omega-3 and Omega-6 polyunsaturated fatty acids). Their omega-3:omega-6 ratio is higher than in terrestrial sources and could be used as nutraceuticals for their anti-hypercholesterolemic, antioxidant, anticancer, antidiabetic, antihypertensive, and anti-inflammatory activities (Chan & Matanjun, 2017; Kumari et al., 2013). Vitamins and Minerals Rhodophyceae species contain important macrominerals, like potassium, sodium, calcium and magnesium (6,1–21,9 g K/100 g d.w., 1,8–8,1 g Na/100 g, 0,2–0,9 g Ca/100 g d.w. and 0,2–0,5 g Mg/100 g d.w.) (Baghel et al., 2014; Kumar et al., 2011; Matanjun et al., 2009). In the group of trace minerals, iron was found to be the most abundant, followed by zinc, copper, and selenium. They could be potential ingredients for functional foods, providing a supplementation in deficitary elements in diets (Nunes et al., 2018). Red seaweeds also contain vitamins, with 2,1–2,7 mg pro-vitamin A/g, 0,05–1,54 mg vitamin B2/g, 3,8–4,8 mg vitamin B6/g, 0,4–1,0 mg vitamin B9/g, 2,5–501 mg vitamin C/ 100 d.w., 1,34 mg vitamin E/g d.w. and 4,61 µg α-tocopherol/g d.w.(Álvarez-Viñas et al., 2019; Chan & Matanjun, 2017). Indeed, vitamins from marine sources have been already used for the enrichment of functional foods (Šimat et al., 2020). Phenolic Compounds Red seaweeds contain a variety of phenolic compounds, including natural phenolics like phenolic acids and flavonoids, as well as specific marine-derived compounds, such as phlorotannin and bromophenol. These compounds are known for their powerful antioxidant properties. Thus, phenolic acids reported in red algae are coumaric acid, caffeic acid, salicylic acid, hypogallic acid, and chlorogenic acid (Carpena et al., 2023). 30 carrageenan disaccharides into less sulphated carrageenans (Souza et al., 2023). Figure retrieved from: Souza et al., (2023). I-carrageenan is essentially a homopolymer of G4S-DA2S disaccharide units, containing a very low amount, of the order of 5 mol %, of G4S-DA disaccharide units. K-carrageenan is somewhat more heterogeneous, although still practically a homopolymer, with up to 10 mol % of G4S-DA2S disaccharide units, discontinuous with blocks of disaccharide units, G4S-DA (Hilliou, 2021; Souza et al., 2023). More simply, these carrageenans consist of polysaccharide chains containing both κ and ι units, as represented in Figure 3, ranging from nearly pure I-carrageenan to nearly pure K-carrageenan (Campo et al., 2009). Within the more heterogeneous carrageenans, the hybrids, K2-carrageenan has a polymeric structure (van de Velde, 2008), with sequences of G4S-DA (which make up 45-80 mol % of the hybrid chain) and G4S-DA2S (which represent 20-50 mol % of the chain) (Hilliou, 2021), hence its definition in the industry as kappa-2 or weak kappa (van de Velde, 2008). So, hybrid K2-carrageenan are copolymers made of blocks of κ and ι with various lengths and random distribution in the chain (Guibet et al., 2008). The block size along the K2 chain, varies from chain to chain, and its distribution in the chain is random (statistic copolymer) and specific to the seaweed biology that produces it, as well as the extraction process. So, K2-carrageenans are statistic block copolymers, with polydispersity both in the chain and in block size (Campo et al., 2009; Souza et al., 2023). Figure 3. Schematic representation of the structure of carrageenans (K, I and hybrids). The κ -units are represented in blue, the ι-units in red and the biological precursors (nu/mu) in green. 2.2.3 Extraction 31 Carrageenans are traditionally extracted from C. crispus wild populations in Canada, Ireland, Portugal, Spain and France, and from Gigartina collected in South America and Southern Europe. However, the growing demands of carrageenans motivated the establishment of macroalgae farming systems, with Eucheuma sp . in Philippines (Carpena et al., 2023; Hedberg et al., 2018) becoming the major producer worldwide and spreading macroalgae cultivation along other Asian countries, promoting the production of Porphyra sp. (nori), Kappaphycus alvarezii (rich in K) and Eucheuma denticulatum (rich in I) (Carpena et al., 2023). The current extraction processes are slow and complicated, involving several steps. Furthermore, it’s quite difficult to optimize and control the production of carrageenan, due to its unique intrinsic properties, which are highly dependent on the origin of the seaweed, the geographical location and the harvesting season (Abdul et al., 2018). Normally, carrageenan industrial extraction processes are divided into drying and milling, alkaline pre-treatment, hot water extraction, precipitation and drying (Souza et al, 2023). Native extracts, without alkaline treatment, generally exhibit poor or absent gelling properties. In contrast, an alkaline treatment at higher concentrations and extended durations results in carrageenan with significantly enhanced gel quality (Azevedo et al., 2015). So, an alkaline treatment is usually performed prior to extraction, but can also be done after, to reduce the number of sulphated groups (or precursors) in K2-carrageenan. This alkaline process can be adjusted to each seaweed (Abdul et al., 2018; Souza et al., 2023). Azevedo et al., (2015) when studying the extraction of K2-carrageenan from two algae, C. crispus and Ahnfeltiopsis devoniensis , observed that excessive KOH concentration promotes the depolymerization of K2 and, consequently, the elasticity of the gel is reduced (see section 2.2.5). The previous study also perceives that after three hours of the alkaline pre-treatment and one hour of natural extraction at 90 ºC, yields for C. crispus achieved with NaOH are larger than those achieved with KOH (45-70 % against 35-60 %), whereas a reverse trend is observed for A. devoniensis (10-20% against 1530%) (Azevedo et al., 2015). Using the same protocol Azevedo et al. (2013) shown that for M. stellatus yields obtained with NaOH were larger than those achieved with KOH (40 - 50% against 30 - 43%), in agreement with results obtained with C. crispus . So, for this two algae NaOH requires lower concentrations and shorter pre-treatment durations compared to KOH to isolate K2 with optimal gel properties, indicating that NaOH is more effective in converting the precursors into κ and ι-carrageenan (Azevedo et al., 2015). The K2 extraction process usually begins by dispersing the algae in hot water (Abdul et al., 2018) It has recently been demonstrated that the solubility of hybrid carrageenans in water is strongly dependent 32 on the type of cations present in the algae after being harvested, the time of contact with the water and the temperature (T) used during extraction (Azevedo et al., 2015). In order to achieve optimal yields, industrial carrageenan extractions are known to be performed at 80 ºC for 6 h. Additionally, it has been reported that the composition and rheological behaviour of carrageenan are different when extracted at different temperatures, for example between 60 ºC and 90 ºC (Bahari, Moelants, Wallecan, et al., 2021). As a rule, a higher T produces a less sulphated K2, however, there are exceptions, e.g. for Gymnogongrus tenuis , a less sulphated carrageenan was obtained in cold water than in water at 80 ºC (Perez Recalde et al., 2016) After extraction, the solid residues (salts formed from cations of the alkaline treatment and from the seaweeds) are separated from the K2-rich solution by filtration or centrifugation. K2 is then recovered from the solution, usually by precipitation with alcohol or with KCl (Abdul et al., 2018). Alternatively, K2carrageenan not subjected to alkaline treatment can be recovered in film or powder form by evaporating water from solutions with high K2 concentrations or by freeze-drying. However, this method is suitable only for K2 with high κ content and sufficient salts to form a strong gel. Finally, purification of K2 can be achieved using enzymatic treatments, among other methods (Souza et al., 2023). 2.2.4 Gel Properties The capacity to form gels is unique to carrageenans that have the ability to form ordered helical structures, being gelation one of the several possible consequences of helix formation (Stephen et al., 2006). Gel formation mechanism The mechanism of gel formation involves the coil-to-helix transition, obtained by cooling hot carrageenan aqueous solutions, followed by the aggregation of carrageenan helices, resulting in a threedimensional network. This mechanism is widely recognized in literature, but this was recently challenged by Westberry et al., (2024) who proposed that the gel formation process is more intricate, involving cooperative interactions and the presence of pre-existing helical structures in the disordered state. This new understanding could prompt a reconsideration of the mechanisms behind carrageenan gelation and related polysaccharides. As for the structure of the network that permeates the volume and provides elasticity is a topic on which there is little agreement among authors. There are studies that report the 33 formation of single helices and others that support the formation of the double helix (Hilliou, 2021). Thus, several models have been proposed trying to describe the structure of the network. Recently, it was possible to visualize by atomic force microscopy (AFM) the primary (coils), secondary (helices) and super helical structures of solutions and gels. For K gels, rod-shaped helices were visualized, while more curved structures were reported for, I gels (Hilliou, 2021). Furthermore, in the presence of K⁺, it was possible to observe quaternary structures of stacked helices linked by helical branching to other domains in K gels. However, for I gels, independently of the type of salt present, the single helix mesh was always observed. Also, the formation of networks at the super helical level, for K gels results in hard gels, in contrast to I gels, which are softer (Hilliou, 2021; Souza et al., 2023; Stephen et al., 2006). The study of K2 gels is still under way. However, the few publications with AFM images suggest that the helices and structure of K2 are similar to those of K, with slightly more branching and a morphology less similar to a “rod”. According to Souza et al. (2023) the mechanism of formation of K2 gel resembles that of K+I mixtures, essentially with coil-helix transitions, independent of each type of block and its aggregation. Nevertheless, it’s indicated that these similarities do not help in the identification of the nature of the aggregation/structuring and even less in understanding the origin of the elasticity of K2 gels (Souza et al. 2023). As K2 gels are different from K+I mixtures, even with a similar G4S-DA (κ) molar content, under similar gelling conditions (total carrageenan concentration and ionic strength), in K+I mixtures, I can be separated from K under specific saline conditions, whereas in K2 this separation is not possible. K2 is capable of adopting helical conformations independently of its κ molar fraction, but there is still a long way to go until a complete understanding of the interaction between such macromolecular structures and the properties of the K2 gel (Souza et al., 2023; Stephen et al., 2006). Additionally, there is no water syneresis in K2 gels, at least in the literature reviewed so far. Thus, the studied K2 gels may be a valuable alternative to the reported mixtures of commercial K and I gels, where water syneresis occurs during gel formation for systems with ionic strengths above 0,1 M. Namely, in applications where an ionic strength as high as 1 M is required, K2 will successfully replace commercial K+I mixtures as syneresis-free gels with similar elasticity are formed (Torres, Azevedo, et al., 2016). Presence of salts Since carrageenans are polyelectrolytes, they are highly sensitive to the composition of the salts present in the aqueous medium (Stephen et al., 2006). So, it is known that it is possible to adjust some gel properties, such as elasticity, yield stress and thermal stability, by controlling the amount and type of 34 salt added (Stephen et al., 2006). K-carrageenan is cation-specific, meaning its gel stiffness and brittleness depend on the presence of specific cations, such as K⁺ or Ca²⁺, which stabilize its helical structure (Hilliou et al., 2006). In solutions with KCl or CaCl₂, K-carrageenan forms strong, stiff, and brittle gels, whereas I-carrageenan, which lacks this specificity, produces much weaker gels regardless of the cation. Thus, the contribution from ι blocks to the gel structure and elasticity in K2 is actually masked by the contributions from κ blocks, when in the presence of the previous cations (K⁺/Ca²⁺). However, in the presence of NaCl, K forms weaker gels, and thus it is easier to access its contribution in K2 gels. As expected, similar results were obtained for the K2 gel, in which the weakest (and most stable) gels were obtained with NaCl, and the strongest for CaCl2. When no salt is used, K2 can show a gel behaviour, but large concentrations and low temperatures are necessary (Torres et al., 2018). Rheology Studies of rheological behaviours, which focus on the dynamic storage/elastic and shear loss moduli (G’ and G’’, respectively) and the yield stress, are relevant for the industrial applications of carrageenans, but also to better understand the gelation mechanism (Souza et al., 2023). According to Van de Velde (2008), in a mixed K⁺/Ca²⁺ solution, the gel elastic modulus (G’) gradually decreased with the decreasing fraction of κ units, indicating that G’ for K2 gels does not reach a maximum at any specific K⁺ concentration. This reflects the intermediate nature of K2 hybrids, characterized by a continuous transition in gel stiffness rather than a distinct peak. However, the gelation temperature (Tg) of K2 hybrids with more than 50 mol% κ units remains independent of composition in the presence of KCl but becomes highly sensitive in NaCl solutions, increasing with higher κ content and decreasing with lower κ content (Souza et al., 2023). Additionally, there is also a strong dependence of the gel’s melting temperature (Tm) on both the concentrations of K-carrageenan in K2 and NaCl (Torres, Azevedo, et al., 2016; Torres, Chenlo, et al., 2016). 2.2.5 Molecular Mass, Mw The molecular mass (Mw) of K2 carrageenans is a complex characteristic influenced by extraction methods, temperature and the specific chemical structure of the carrageenan itself. The effect of molecular mass on the yield stress and G’ of the gel was determined, for K-carrageenan, with different values of polymer concentrations and salt concentrations by Rochas et al., (1990), that concluded that 35 the yield stress is directly proportional to the molecular mass, but not very sensitive to the salt concentration. In contrast, the G’ increased steadily with Mw, up to a critical value (Mc) of 1,8·105 g/mol, independently of the polymer concentration and ionic content. Beyond this limit, the G’ remained constant and independent of the Mw, but very sensitive to the salt concentration (Rochas et al., 1990), as seen in Figure 4. For K2-carrageenan, there is still insufficient information to understand the effect of varying Mw and whether Mc depends on the chemical composition in κ and ι of K2. Figure 4. Illustration of the variation of the elastic modulus (G’) with molecular mass (Mw) for K-carrageenan. It stabilizes from Mc, regardless of the 3 different polymer concentrations (5, 10, 20 g/L). Adapted from: Rochas et al., (1990). Impact of Extraction Parameters The molecular mass of isolated K2 samples is significantly influenced by the milling process prior to biopolymer extraction. Grinding seaweeds into smaller particles increases the yield of K2-carrageenans during water extraction, resulting in compounds with higher molecular masses. Consequently, larger molecular masses are obtained from powders with smaller particle sizes, while smaller molecular masses are associated with larger particle sizes. Furthermore, the polydispersity index (PDI), represented by the Mw/Mn ratio (where Mn is the number-average molecular weight), tends to increase with Mw (Torres et al., 2016). In the previous reference it was observed that differences in K2 molecular mass were likely due to larger K2 molecules experiencing greater diffusion resistance through the seaweed particles during extraction. In contrast, powders with smaller particle sizes resulted in a higher proportion of large K2 G’ 36 molecules, likely due to increased surface area and enhanced accessibility of precursors to the solvent. These findings suggest that particle size distribution and diffusion characteristics significantly impact the yield and molecular composition of the extracted fractions (Torres, Chenlo, et al., 2016). Molecular mass is also highly influenced by the extraction parameters used during the recovery of carrageenan. It is well known that stronger and longer alkaline pre-treatments tend to reduce the chain size of K2, resulting in a lower Mw (Souza et al., 2023; Van De Velde et al., 2005). On K2-carrageenans studies showed that NaOH is faster than KOH in converting the biological precursors (mu e nu) into the disaccharide units κ and ι (Azevedo et al., 2013, 2015). So, this efficiency is reflected in the Mw of the resulting K2, as shorter alkaline treatment times are required to achieve the desired levels of sulphated disaccharide units (Velde et al., 2005; Souza et al., 2023). The temperature variations can also lead to changes in Mw, resulting in different types of gels. During the drying step, in the case of M. stellatus , higher drying temperatures led to a lower Mw of K2carrageenan (Moreira et al., 2016). But for K2 extracted, from Gymnogongrus tenuis, using cold water, in the extraction step, resulted in a different Mw profile compared to a more sulphated hybrid carrageenan recovered at 80 °C, which does not qualify as K2-carrageenan. Although, higher temperatures generally increase yield, excessive heat can lead to hydrolysis, causing degradation. Therefore, it is important to carefully control the extraction temperature to prevent excessive breakdown of the carrageenan (Bahari, Moelants, Wallecan, et al., 2021; Moreira et al., 2016; Souza et al., 2023). Carrageenan hydrolysis can also occur in low pH conditions (Gustaw & Mleko, 2003) or under high salinity conditions, such as in the presence of NaCl or KCl. High salt concentrations can disrupt the intermolecular interactions that maintain carrageenan structure, making it more susceptible to hydrolysis, particularly when combined with high temperatures or acidic conditions. However, studies on the effects of salts on carrageenan extraction remain surprisingly limited (Bahari, Moelants, Wallecan, et al., 2021). Toxicity Understanding the Mw of K2-carrageenans is essential for its industrial applications, particularly in food and cosmetic industries where textural properties are critical. The ability to manipulate Mw through extraction methods can lead to tailored products that meet specific consumer needs, with the desired textures and stability in products. For example, food-grade carrageenan typically has a molecular mass greater than 100 kDa, with commercial products ranging from 200–800 kDa (Torres, Flórez-Fernández, et al., 2019). Notably, carrageenan is neither degraded nor absorbed in the gastrointestinal tract, but it 37 should not contain low molecular mass fractions, as these can have toxic effects, including gastrointestinal irritation and potential carcinogenic properties at high doses (Torres, Kraan, et al., 2019; Weiner, 2016). Further studies are necessary to explore its impact on digestive processes, the colon microbiome, and inflammation, especially in predisposed populations (Wu et al., 2016). To despite the previews claims in a review article, Bixler, (2017) emphasize that carrageenan is approved for food use by major regulatory agencies worldwide, including the example of its recent inclusion in liquid infant formula, that highlights its safety in sensitive applications, and that there is no in vivo evidence to support the previous claims. Thus, while regulatory bodies affirm its safety, public scepticism fuelled by misinformation remains a significant hurdle for the industry. In resume, there is a recognized need for more studies focusing on the optimization of extraction parameters to better understand how they affect the Mw and chemical structures of K2-carrageenans. In particular, there is no published results (such as those illustrated in Figure 4) on the effects of Mw on the gel properties of K2-carrageenans, and this could lead to improved applications and functionalities of K2carrageenans in various industrial contexts. 2.3 Sequential Extraction The current industrial extraction routes in the carrageenan sector have several weaknesses, primarily due to their high consumption of time, energy and water. Additionally, large amounts of chemical reagents and solvents are required to achieve optimal yield and quality. The pollutants and effluents generated from chemicals used during extraction and alkaline treatment, such as KCl, KOH, and NaOCl, pose significant risks to health and the environment, necessitating strict control measures (Abdul et al., 2018). In addition, the alkaline treatment step, in particular, is a relatively harsh and destructive process for the algae, causing the breakdown of the tissue matrix and the efficient solubilization of nearly all the carrageenan. The residual algal biomass, still rich in organic components, is generally unrecoverable and wasted after this step. So, shortening the time of the alkaline step, while still producing carrageenans of high rheological quality would undoubtedly be of interest to the industry (Vauchel et al., 2008). Consequently, greener extraction methods that use less energy, reduce water consumption, and employ more environmentally friendly solvents are emerging. Microwave-assisted extraction (MAE) (Álvarez-Viñas et al., 2023) and Ultrasound-Assisted Extraction (UAE) (Maia et al., 2023) have shown the best results in terms of extraction yields and K2 gel properties, with a shorter extraction time and ecological impact (Kes 38 et al., 2017). However, MAE and UAE are difficult to apply in industries and expensive (Sugiono et al., 2019). Another hypothesis involves using a twin-screw extruder-assisted extraction, which offers several advantages: it requires shorter processing times, reduces the amount of solvent and reactants needed, generates minimal waste, and is safe and suitable for industrial applications (Vauchel et al., 2008). Additionally, the current batch industrial process for carrageenan extraction is limited to producing only one polymer per cycle, leaving many valuable compounds unrecovered and wasted, so the full potential of the algal biomass is not being utilized. The global carrageenan industries process 202 500 dry tons of carragenophytes annually to produce 65 000 tons of carrageenan (Penuela et al., 2018) and the remainder is lost as waste. With the increase of carrageenan production expected in the coming years, the valorisation of the waste fractions becomes necessary. 2.3.1 Biorefinery Strategies for the Valorisation of Red Seaweeds A succession of extraction steps can lead to the sequential separation of products of interest, from the same biomass, maximizing its value and generating fewer residues, while maintaining the focus on the production of phycocolloid. For carragenophytes, a biorefinery approach should prioritize carrageenan extraction as the primary objective, given its well-established and industrialized production process (Álvarez-Viñas et al., 2019; Balina et al., 2017; Torres, Kraan, et al., 2019). Efforts to valorise sides streams and utilize the entire red algae biomass through a biorefinery approach are increasingly being discussed. Various models have been proposed for seaweeds to produce a wide range of products, typically concluding with the extraction of phycocolloids, after all other valuable components have been removed. In fact, biorefinery concepts have already been applied to M. stellatus industrial waste from conventional extraction and have demonstrated that K2-carrageenan and antioxidant compounds can be further extracted from the waste (Bianchi et al., 2022). Maia et al. (2023) developed a 3-step sequential extraction using UAE to recover pigments, proteins, and carrageenans from Chondrus crispus . The study reports the presence of chlorophyll-a and carotenoids with mean values varying from 289,2 and 432,2 µg/g extract d.w., to 39,9 and 59,9 µg/g d.w., respectively. Other fractions obtained through UAE contained proteins and carrageenans yields in the ranges of 3,6–41 g/100 g and 29,7–36,1 g/100 g, respectively. Moreover, Bahari et al. (2021) proposed a method to extract both phycoerythrin and hybrid 39 carrageenan from the same species, beginning with the extraction of phycoerythrin under mild conditions. Their findings showed that at an extraction temperature of 22 °C, reducing particle size significantly enhanced phycoerythrin concentration (from 0,26 mg/g in >2000 µm particles to 2,30 mg/g in <50 µm fractions). In contrast, carrageenan yield only slightly increased (from no recovery in >2000 µm particles to 2,1 g/kg in <50 µm), remaining much lower than the yields obtained at higher temperatures of 45 ºC (5,4 g/kg) and 90 ºC (9,9 g/kg). These results suggest that phycoerythrin extraction is surface-area dependent, whereas carrageenan extraction relies more heavily on temperature. A sequential extraction approach has also been tested with Solieria filiformis to obtain a nutrient-rich extract, PUFA-rich lipids, and pure I-carrageenan, using MAE. This method demonstrated the potential for maximizing yields while generating only 6,3–10,4 % of residues from the initial biomass, a significant reduction compared to the residues generated through direct extraction of each product, which were 15 times higher (Penuela et al., 2018). In a study by Bahari et al. (2021), the extraction of K2-carrageenan from C. crispus was evaluated using a sequential approach under different times and temperatures, without the use of alkaline pre-treatment. This elimination aimed to explore a cleaner extraction process, though it allowed precursor units (mu and nu) to remain in the carrageenan, reducing its gelling properties. Besides, it has been shown that carrageenan’s composition and rheological behaviour can vary significantly depending on the extraction temperature and duration. Knowing that the conventional industrial processes, carrageenan is typically extracted at 80ºC for 6 hours, the previous study investigated two types of extraction procedures: the first was a two-step process, beginning with an initial wash (0,5 hours) and extraction of the seaweed at room temperature for 8 hours, followed by a second (sequential) extraction at a specific temperature (ranging from 22 to 90ºC) and duration (from 2 to 8 hours); and the second method involved a direct extraction without an initial wash, conducted at a specific temperature and duration. Results indicated a linear correlation between the yield of carrageenan-rich precipitate (CRP) and the combination of temperature and time, suggesting that carrageenan solubility and leaching increase gradually with rising energy input. Additionally, the study found that removing endogenous salts improved the yield of hybrid carrageenan at lower extraction temperatures. The effects of sequential extraction methods on the functional properties of agars and porphyrans derived from red seaweeds, with the aim of improving extraction yields and valorising byproducts, was investigated by (Gomes-Dias et al., 2024). The study evaluated three extraction techniques: cold-water extraction (CWE), ethanolic extraction (EE), and alkaline extraction (HAE) — both individually and in combination, to determine their impact on the quality and yield of the extracted 46 was centrifuged at 8 000 g for 10 minutes, and the liquid fraction, containing carrageenan and salts, was transferred to a plastic container and dried at 55 ºC overnight. The next day, the dried sample (of the three replicas) was weighed to determine the total mass after HAE. Then, from this total, a portion (e.g., 5 grams) (mass after HAE used for the precipitation), was dissolved in water to form a 2 % (w/v) carrageenan solution. This solution was then subjected to ethanolic precipitation to remove excess salt and isolate the carrageenan (Azevedo et al., 2013). The precipitated carrageenan was transferred to a container and dried again in an oven at 55ºC overnight. The following day, the dried precipitate was weighed (mass precipitated) to obtain the yield, YY, Equation (5). (mass precipitated · total mass after HAE) (total algae mass · mass after HAE used for the precipitation) (5) 3.1.8 Characterization of hybrid carrageenans Gels preparation and viscoelastic characterization K2 extracts 2% (w/v) were dissolved in solution with 1 M NaCl and stirred vigorously for 1 h at 80 ºC. The concentration and ionic strength were specifically selected to obtain gels with sufficient rigidity at 25 ºC, without water release, and to maintain a single cation (Na⁺) to facilitate gel formation, since the alkaline extraction was carried out with NaOH (Azevedo et al., 2014). After allowing the solutions to cool for 24 h, two possible outcomes were observed: a) gel formation or b) no gel formation. a. Gel formation) The samples that gelled were reheated to 85 ºC and the resulting hot K2 solutions were directly transferred into the pre-heated (80 ºC) Couette accessory (CC10) of a Paar Physica MCR300 rheometer (Anton Paar, Austria). The geometry surface was covered with dodecane oil to prevent water evaporation. The samples were then cooled to 25 ºC within 5 500 seconds (≈1,5 h). During the cooling of the solution a small amplitude oscillatory shear was applied (with 0,1 % strain at a ω=1 Hz, the ω being the frequency) to probe the temperature evolution of linear viscoelastic properties such as tan δ=G’’/G’. The temperature where tan δ =1 was defined as the gel setting temperature, Tg, as seen in Figure 6 a). Following the cooling and gel setting, at a 25 ºC a frequency YY = 47 sweeps was performed with a 0,1 % strain, which ensured the measurement of equilibrated gel’s mechanical spectra in the linear regime and the determination of the G’ at 1 Hz, label as G0 (Figure 6 b)). A shear strain (ɣ) sweep from 0,01 to 1000 % with a frequency of 1 Hz was then made to verify that the produced frequency sweep and G0 were acquired within the linear regime of elasticity (Souza et al., 2011) and to characterize the gel behaviour under large deformation, as shown in Figure 6 c). Figure 6. Example of the experimental protocol followed for the rheological characterization of the gels, in the Paar Physica MCR300 rheometer (Anton Paar, Austria). b. No gel formation) The solutions were placed into a plate-plate geometry (40 mm diameter) of a stress-controlled rotational rheometer (MCR 302, Anton Paar) at 25 ºC. Each sample was equilibrated during 300 seconds by applying an oscillatory strain of 10 % at 1 Hz and 25 ºC. This step was followed by a ω sweep from 100 to 0,01 Hz, performed with a strain of 50,3 %, in view to record the mechanical spectrum of the solution and collect the G’ at 1 Hz, label as G1 (as seen in Figure 7 a)). Then, a shear rate (ɣ) sweep ranging from 1000 s-1 to 1 s-1, immediately followed by sweep from 1 s1 to 1000 s-1, were performed to measure the shear rate dependence of the apparent shear viscosity, η, to measure the flow curve of the solution and assess any possible thixotropic behaviour related with carrageenan aggregation with time, shown in Figure 7 (b) and c)). G’= G’’ 48 Figure 7. Example of the experimental protocol followed for the rheological characterization of the liquids, in stress-controlled rotational rheometer (MCR 302, Anton Paar). Spectroscopic analyses The Fourier Transform Infrared Spectroscopy (FTIR) method was used for the qualitative chemical characterization of the extracted K2. The FTIR analyses were performed on a Perkin-Elmer Spectrum 100 FTIR spectrometer equipped with an Attenuated Transmission Reflectance (ATR) accessory. The spectra were obtained in a range of 4000 to 600 cm−1 with 16 scans per sample and a resolution of 1 cm-1. The assignments of the FTIR spectra were mostly based on the previous work of (Van De Velde et al., 2004) (see Table 5). Nuclear Magnetic Resonance (NMR) spectroscopy was performed using a 400 MHz Avance III spectrometer (Bruker) at 70 ºC to limit the line broadening caused by the viscosity of the 1 wt.% carrageenan solutions in D2O. Indeed, the sample solutions were ultra sonicated during hours (using a DCG-300H bath, MCR Ltd.) to lower their viscosity prior to NMR characterization. Chemical shifts (ppm) were referenced to the D2O lock signal (4,41 ppm) and further adjusted to match the chemical shift for κ (5,09 ppm), as prescribed by van De Velde et al., (2004). The molar fractions (mol %) of the carrageenan repeating units are calculated as the integrated intensity of the corresponding 1H-NMR peak (see Table 6) over the sum of the integrated intensities of all assigned carrageenan anomeric protons (Van de Velde et al.,2004). The pyruvic acid acetals and floridean starch are also detected in the 1H-NMR spectra by the methyl proton resonances with chemical shifts, relative to DSS (an NMR standard), of 1,44 ppm for the 49 pyruvic acid and 5,35 ppm and 4,98 ppm for floridean starch (Van De Velde et al., 2004). The NMR spectra were produced by the chemical analyses service at the department of chemistry of the University of Minho. Table 5. Identification of carrageenan types by infrared spectroscopy (adapted from (Chopin & Whalen, 1993)). The intensity of the observed peaks is indicated by symbols: ‘+’ (weak), ‘++’ (moderate), ‘+++’ (strong) and ‘s’ (shoulder peak) Wavenumbers (cm-1) Bond(s)/group(s) Letter Code Type of carrageenan κ mu ι nu L 1240-1260 S=O of sulphate esters - + ++ ++ +++ +++ 970-975 Galactose D + s + s - 930 C–O of 3,6-anhydrogalactose DA + - + - - 845 C–O–SO3 on C4 of galactose G4S + + + + - 825-830 C–O–SO3 on C2 of galactose D2S - - - + + 815-820 C–O–SO3 on C6 of galactose D6S - + - + + 805 C–O–SO3 on C2 of 3,6anhydrogalactose DA2S - - + - - Table 6. Chemical shifts (ppm) of the alfa-anomeric protons of carrageenans referred to DSS as internal standard at 0 ppm (adapted from: Van de Velde et al.,2004) Types of carrageenan Monosaccharide Chemical shift (ppm) κ DA 5,093 Mu D6S 5,238 ι DA2S 5,292 Nu D2S,6S 5,501 50 Molecular mass determination The molecular mass distribution was obtained by size exclusion chromatography and refractive index detection from a service purchased to the Analise Laboratory of the LAQV-REQUIMTE Associate Laboratory, NOVA University of Lisbon. A Waters 600 apparatus coupled to a Waters 2410 differential refractive index detector was used, whereas the size exclusion chromatograph was a PolySep-GFC-P Linear column (Phenomenex, Alcobendas, Spain). This column has been calibrated with pullulan standards (Shodex, Munich, Germany) ranging from 6 300 to 642 000 g/mol. Duplicated measurements of Mw and PDI were performed with 0,1 wt % carrageenan solutions in 0,1 M NaCl at 40 ºC. 3.2 Study of the Molecular Mass 3.2.1 Raw Material The K2-carrageenans utilized in this study were hot-water extracted and then subjected to a 3% (w/v) KOH alkaline treatment by another research group, following earlier results reported by (Azevedo et al., 2013). The extractions were conducted with five commercially sourced algae, generously provided by AlgaPlus® and Cargill®. The resulting hybrid carrageenans were assigned specific codes and had varying chemical compositions to assess the impact of molecular structure on the breakdown of molecular mass: B-87 (4 mol % κ and 96 mol % ι); Gigartina (43 mol % κ, 40 mol % ι, and 17 mol % nu); Mastocarpus (70 mol % κ and 30 mol % ι); BAT 18-09 (56 mol % κ, 36 mol % ι, 4 mol % mu, and 4 mol % nu); and C25 (90 mol % κ and 10 mol % ι). 3.2.2 Reagents The reagents used during this work are described on Table 7, such as the companies that provided those reagents. Table 7. Reagents used during the ultrasonication of K2-carrageenans Reagents Company Potassium chloride Sigma Aldrich Dodecane Sigma Aldrich 51 3.2.3 Ultrasonication of carrageenans and gel test K2-carrageenan samples were dissolved in distilled water with vigorous stirring at 80 ºC for 1 h to ensure complete dissolution, at a concentration of 0,5% (w/v), once is know that at lower concentrations the Mw breakdown of the polymer is faster (Rochas et al, 1990). Following this, the carrageenan solutions underwent ultrasonication for various durations in an ultrasonic bath (DCG-300H, MCR Ltd.), operated at 40 kHz and an ultrasonic power of 400 W in pulsed mode at 80 ºC to mechanically reduce their molecular mass by cavitation, rather than by hydrolysis route which could affect the hybrid carrageenan composition. Ultrasonication was applied for various durations (1, 2, 3, 4, 12, 14, 16, and 20 h), generating distinct samples at each time interval. A gelation test was performed on each sample post-ultrasonication by preparing 1 % (w/v) carrageenan solutions in 0,1 M KCl. The solutions 0,5 % (w/v) were first evaporated to reach the prior concentration, followed by the addition of KCl in its salt form. These were heated to 80 ºC for 1 h and then cooled to room temperature for 24 h to induce gel formation (Azevedo et al., 2015). As a control, a non-sonicated sample (0 h) was included, and all the carrageenan samples successfully formed gels at 0 h. The gelling properties of the samples following ultrasonication were compared, revealing that some samples lost their ability to gel after 4 hours of treatment, while others remained gelled until 20 hours. The ultrasonication duration of a carrageenan solution stopped when an aliquot sample remained liquid after the gel test. 3.2.4 Rheology After the gelation test, the K2-carrageenan samples subjected to different ultrasonication times, as well as the non-sonicated control sample, were characterized by rotational rheometry to compare the gelling behaviour across the samples. The liquids were characterized as described in section 3.1.8 (Gels preparation and viscoelastic characterization). As for the gel, a new geometry and rheometer was used – to accommodate for the small number of available samples. A different plate-plate geometry (40 mm diameter) of a stress-controlled rotational rheometer (MCR 302, Anton Paar) was used, but the experimental protocol (Figure 8) closely resembles the previous one (Figure 6). It differs in the control of gel thickness, with the normal force set to 0 N during cooling. A new characteristic is evaluated: TON. This temperature is measured with more precision than the inflection point, Tg (see Figure 8.a.1)). TON can be identified from the Gap (mm) reduction, which indicates that the sample is transitioning from a liquid (where the temperature dependence of the gap is linear due to the thermal expansion of the shearing geometry) to a gel state (with the contraction of the sample’s volume), as illustrated in Figure 52 8.a.2). The mechanical spectra (Figure 8 b)) and the shear strain test (Figure 8 c)) were performed as described before (in section 3.1.8). Figure 8. Example of the experimental protocol followed for the rheological characterization of the gels, in the stress-controlled rotational rheometer (MCR 302, Anton Paar). 3.2.5 Spectroscopic analyses The FTIR method was employed for the qualitative characterization of the chemical structure of K2-carrageenan, comparing the non-sonicated sample (0 h) with the sample subjected to the longest ultrasonication time. This analysis aimed to verify whether any modifications occurred in the chemical structure of the carrageenan after the ultrasound. The experimental protocol followed is detailed in section 3.1.8 (Spectroscopic Analyses). 3.2.6 Molecular mass determination To assess the molecular breakdown, the molecular mass of the carrageenans prior to and during ultrasonication was determined. The experimental protocol followed is detailed in section 3.1.8 (Molecular mass determination). 53 3.3 Statistical analysis Statistical analysis was performed to identify significant differences using one-way ANOVA in Origin version 2024b (Learning Edition). Multiple comparisons were evaluated through Tukey's test at a 95 % confidence interval. For each seaweed species and carrageenan sample, an analysis was conducted to assess whether the sequential extraction and ultrasound methods produced significant differences (p < 0,05) in the yield of extracted fractions, the Mw and the PDI. Results are presented as means ± standard deviation, with a minimum of two replicates conducted unless otherwise specified. 54 4. RESULTS AND DISCUSSION 4.1 Sequential Extraction The sequential extraction (SE) process enables the fractionation of pigments and K2carrageenans over time, isolating fractions with distinct chemical and rheological properties. By analysing yields, chemical composition, and rheological behaviour across extraction stages, this study assesses the impact of SE and extraction time on the functional properties of K2-carrageenans. These findings are crucial for optimizing extraction time to screen for the potential of extractive extrusion, and tailoring carrageenans for specific industrial applications. First, to validate SE as a viable alternative to conventional batch extraction for K2-carrageenans, the yields of fractions recovered at each step are analysed in relation to the known compositions of the studied seaweeds. Subsequently, the chemical structures of the carrageenans obtained through SE are discussed, followed by an in-depth assessment of their rheological properties. 4.1.1 Cold Water extraction (CWE) The first extraction consists of an overnight cold water extraction. The compound extracted is a known pigment of the red algae, but also a protein, R-phycoerythrin (R-PE) (Bahari, Moelants, Kloeck, et al., 2021). This extraction does not have a control, as it is the first extraction and therefore not influenced by any prior extraction. The absorbance values at specific wavenumbers of the various liquid extracts (triplicated samples) can be consulted Appendix A. Comparing the R-PE yields shown in Figure 9, specifically, 0,343 ± 0,058 mg R-PE/g d.w. for M. stellatus , and 0,214 ± 0,062 mg R-PE/g d.w. for C. crispus , with literature values of 1,99 mg R-PE/g d.w. for M. stellatus and 0,53 mg R-PE/g d.w. for C. crispus (Nguyen et al., 2017; Pina et al., 2014), it is evident that less than half of the R-PE reported in the literature is extracted from the algae studied here. As for G. pistillata no values are available in the literature to compare with the 0,307 ± 0,049 mg R-PE/g d.w. shown in Figure 9. Furthermore, C. crispus shows comparatively lower R-PE yields than the other two algae in this study and in the literature. The low R-PE yield observed in this study may be attributed to non-optimized extraction conditions. For instance, Nguyen et al. (2017) achieved significantly higher yields for M. stellatus by using a different method involving phosphate buffer (20 mM, pH 7,1) and xylanase to degrade the algal cell 55 wall, yielding up to 1,99 mg R-PE/g d.w. Further studies should consider varying the algae used within M. stellatus and the other species, to assess seasonal and geographical impacts on R-PE yield, as R-PE content may differ between winter and summer. It is possible that this extract does not only contain R-PE, but also salts from the seaweed, as well as sulphated carrageenans (e.g. L-carrageenans), as some studies reveal that it is possible to extract carrageenans at 25 ºC, at extended times (Bahari et al, 2021). So, the liquid extracts were analysed further and discussed in section 4.1.5. Influence of the sequential steps. Figure 9. Extraction yield (mg/g d.w.) of the phycoerythrin, R-PE, with standard deviation error bars, for the three different algae ( Mastocarpus stellatus, Chondrus Crispus and Gigartina pistillata ), from the overnight (16h) cold extraction, first step of the sequential extraction. 4.1.2 Ethanolic Extraction (EE) The second extraction, based on ethanol, showed varied outcomes for the algae, the extraction yield of chlorophyll-a (chloro-a) and carotenoids (carot), are shown in Figure 10 and Figure 11, respectively. Details on the values of each triplicated sample and absorbance is given in Appendix B. For M. stellatus , C. crispus , and G. pistillata , the chlorophyll-a yield showed an increase or statistical similarity yield across extraction times (10 min to 1 h), when analysing separately both extraction types (sequential and control). In M. stellatus , the 1 h SE extract produced a lower yield than the 1h control, but for smaller times (10–30 min) the yield was similar. Conversely, C. crispus exhibited a slight increase in chlorophyll-a yield in SE extractions, likely due to the initial CWE enhancing pigment diffusion. Similarly, G. pistillata showed improved recovery with the 1 h SE extraction compared to shorter 62 Table 9. Peaks associated with carrageenan in the infrared spectra of different alga across specific wavelength regions. The intensity of the observed peaks is indicated by symbols: “-” (no peak), “+” (weak), “++” (moderate), and “+++” (strong), with “no data” representing samples that were not subjected to FTIR analysis. The yellow columns are the ones related to κ or/and ι-carrageenan, and the white columns relate to mu and/or nucarrageenan 4.1.5 Influence of the sequential steps The liquid extracts obtained during the initial cold water extraction (CWE) with C. crispus and G. pistillata were analysed to determine carrageenan content, and the presence of non-gelling carrageenan in these extracts was observed in the FTIR and NMR spectra. This is in agreement with Bahari et al. 1240-1260 970-975 930 845 825-830 815-820 805 CWE Sequential 16h - - - - - - - 1 h + + ++ ++ + + + 30 min + - ++ ++ + - + 10 min 1 h + + ++ ++ - - + 30 min + + ++ ++ - - + 10 min 1 h ++ + - + - - + 30 min - + + ++ - + + 10 min 1 h + + ++ +++ - - + 30 min + - - ++ - + + 10 min CWE - 16 h + - ++ ++ - - + 1 h +++ ++++ +++ - - ++ 30 min ++ ++++ +++ - - ++ 10 min +++ ++++ +++ - - ++ 1 h +++ ++++ +++ - - ++ 30 min +++ ++++ +++ - - ++ 10 min +++ ++++ +++ - - ++ 1 h + + ++ ++ - - + 30 min + + ++ ++ - - + 10 min + + ++ ++ - - + 1 h + - - + - - + 30 min + - + +++ - - + 10 min - + + +++ - - + CWE - 16 h + - ++ - + - + 1 h + - ++ + - - + 30 min + + ++ + - - + 10 min + + ++ + - - + 1 h - - ++ + + + - 30 min - + ++ + + + + 10 min - + ++ + + - - 1 h + + + ++ + - + 30 min + - - - - - + 10 min 1 h + + + - - - + 30 min + + + ++ - + + 10 min Mastocarpus Stellatus time Extracted K2-carrageenans Peaks at the wavelengths (cm¯¹) Algae Extract Type Control Sequential HWE Control Sequential HWE Control Sequential HAE HWE Control Sequential Gigartina pistillata no data no data no data no data no data no data Chondrus Crispus HAE Control Sequential HAE Control Sequential 63 (2021), who reported approximately 5 % yield of carrageenan, from C. crispus, following an 8 h extraction at 22ºC, plus an additional 2 h extraction at the same temperature. The previous study confirmed that a small fraction of carrageenan, particularly non-gelling, L-carrageenans, as determined from FTIR, can be recovered under room-temperature conditions. No detectable K2-carrageenan were found in the extract from M. stellatus , as the liquid failed to form a film upon drying. This result strongly suggests that the CWE in M. stellatus has no impact on the recovery of K2-carrageenans. Thus, the total carrageenan yield of nearly 50 % of the previous algae in both SE processes (HWE and HAE) is in harmony with the carrageenan yields reported elsewhere for hours long alkali extraction routes (Azevedo et al., 2013), whereas it is almost twice the yield reported more recently for a HWE performed at 80-90 ºC for a total of 3 h (Sanz et al., 2023). In the case of G. pistillata and C. crispus , both κand ι-carrageenans were identified alongside their precursors, muand nu-carrageenans. The FTIR band at 825–830 cm⁻¹ for G. pistillata is indicative of the substantial presence of nu-carrageenan, corroborated by the ¹H-NMR spectrum. Moreover, the characteristic peak at 5,54 ppm in the ¹H-NMR spectra confirmed the presence of L-carrageenans in these extracts but is not the major component as claimed elsewhere from FTIR spectra (Bahari et al., 2021). Essentially, CWE products from G. pistillata are more sulphated than the sequential and control HWE products. The Mw of the carrageenans extracted through CWE was found to be comparable to those obtained from HAE for the same species, suggesting that the cold-water process is capable of recovering carrageenan chains of similar lengths, but it needs a longer time. However, PDI of the CWE extracts from C. crispus was notably high (4 ± 0,038), reflecting a broad distribution of molecular masses, likely influenced by the mild extraction conditions and the presence of precursors and L-carrageenans. An important observation is that the removal of carrageenan during the CWE could be a contributing factor to the reduced sulphation levels observed in subsequent SE steps. The preferential extraction of more soluble and highly sulphated carrageenans in the CWE may leave behind a carrageenan fraction richer in less sulphated hybrid carrageenans. This hypothesis aligns with the findings of lower sulphate content and enhanced rheological properties in SE extracts. So, the removal of precursors during CWE not only shifts the composition of subsequent extracts but also impacts their molecular characteristics, favouring the recovery of less sulphated carrageenans with improved gelforming abilities. 64 Analysing the HWE results of M. stellatus , FTIR spectra revealed that SE carrageenans shared similar peaks with the controls. However, in the NMR spectra the 1 h SE extract exhibited a higher sulphate content than the control, at 30 min it was the opposite. In the subsequent HAE, alkali extracts (both SE and controls) didn’t show precursors, aligning with expectations since the alkaline medium favours the extraction less sulphated carrageenans. An exception was observed for the 30 min control alkaline extract, where ¹H-NMR identified muand nu-carrageenans, suggesting that prior steps in the 30 min SE alkaline extract may have facilitated the recovery of less sulphated carrageenan in this step. Regarding the impact of the SE on Mw and PDI, water-based M. stellatus SE extracts displayed increased PDI, likely due to prior steps broadening the chain size distribution. The Mw of the 1 h SE water and alkaline extract was higher than its control, while for the 30 min, it was similar. For C. crispus , FTIR spectra showed minimal differences between water-based SE and control extracts. However, ¹H-NMR revealed that all the water extracts contained precursors (less than 16 mol% in total), with the SE extracts having a slightly lower molar content of precursors than controls. Accordingly, gels are obtained with SE HWE extracts in contrast to the liquids obtained with extracts from control experiments, as will be discussed in section 4.1.8 Texturizing behaviour. In the alkaline extraction step, differences were observed in FTIR signals at 930 cm⁻¹ and 845 cm⁻¹ between SE and controls. However, since these signals are common across carrageenans, it’s challenging to attribute them to specific chemical changes caused by SE. Notably, ¹H-NMR showed stronger κ-carrageenan signals in controls, which also contained mu-carrageenans absent in SE extracts. Together with the computed yields, this again suggests that more sulphated carrageenans were extracted during the HWE step, whereas parts of such chains still resist the alkali treatment and are thus extracted during the control experiments. With respect to the molecular mass distribution, the sequential approach in both HWE and HAE has no impact, except for shorter times with HWE and longer times for HAE, where sequential extractions gave longer chains than the control in the HWE and vice-versa in the HAE. The PDI values remained insensitive to the extraction protocols for C. crispus . For Gigartina pistillata , SE products from HWE performed at shorter times contained fewer precursors than the controls. In the alkaline step, SE and control extracts were largely similar. In this alga, SE water extracts consistently displayed higher Mw than controls, suggesting that the SE sequence positively impacted Mw recovery. In alkaline extractions, the Mw of SE and control samples were quite similar. 65 In summary, Mw trends suggest that SE generally contributes to a more consistent recovery of high Mw carrageenans compared to controls, particularly during the HWE step. This effect was more pronounced in M. stellatus and G. pistillata , while C. crispus exhibited more stable Mw across conditions. Combining these observations with FTIR and ¹H-NMR data, it is evident that SE enhances the recovery of less sulphated carrageenans, especially in shorter extraction times. This suggests that the CWE step likely facilitated the leaching of more sulphated polymers which are not found in the subsequent extractions. Furthermore, SE extracts demonstrated superior rheological properties, with higher G’ in gels and higher (or similar) viscosity in liquid states compared to their respective controls. These findings highlight the benefits of incorporating sequential extraction for improved carrageenan quality, particularly in optimizing Mw, sulphation levels, and rheological performance. 4.1.6 Effect of time For this thesis, three extraction times were study: 1 h, 30 min and 10 min. This parameter affects the yield of the extraction as discussed before, but also the chemical composition of the type of carrageenans extracted. For the HWE M. stellatus results, the 1H-NMR reveals that the 30 min water SE K2-carrageenan, had no precursors (mu and nu), whereas 1 h had a 32,13% molar content of precursors. A similar trend is observed in the control samples, where the 1h extraction results in the removal of more precursors than the 30 min extraction (13,64 % molar and 3,84 % molar, respectively). Finally, M. stellatus is not suited for extraction times as short as 10 minutes, once the yield was very low and the extracts that were evaluated didn’t show K2-carrageenan. As for the C. crispus , 1H-NMR reveal that at shorter times (30 min and 10 min) of extraction the SE water extracts had less precursors, which is reminiscent from the results found for M. stellatus . However, the opposite is found for the controls. In the alkaline step, no precursors were detected for the two last algae. And it seems that for C. crispus the 10 min extraction delivers a κricher K2-carragenan than the 1 h, in the SE and controls. For G. pistillata , smaller SE HWE extraction times led to lower precursor contents (see FTIR with less pronounced bands symptomatic of sulphated groups and NMR results), in concordance with the other two algae. In the alkaline step, the chemical composition of K2-carragenans does not seem to be affected by the time, with the exception of a small molar percentage (4%) of nu in the 1h SE alkaline extracts. So, the general trend is that with more time, more carrageenan (see Figure 12) with more sulphate is leached out from the seaweeds during the HWE. Practically, and in view the optimized 66 texturing properties of carrageenans, more extraction time brings benefits when alkaline is used as more carrageenan (see Figure 13) with less precursors are recovered. As for the impact of time in the molecular mass, for M. stellatus , longer extraction times consistently leads to the isolation of longer or similar hybrid carrageenan chains with identical PDI. The same trend is found with C. crispus, except for K2-carrageenans obtained from the sequential HAE where smaller chains are recovered at longer times. Note that this may explain the corresponding small carrageenan yield as smaller chains are more difficult to precipitate. In G. pistillata , SE water extractions had a similar Mw across the time. As for the alkaline SE extracts there is an increase Mw over time, while the PDI maintains consistent. So, the overall trend is that the lower Mw polysaccharides are solubilized first at earlier times. These results also reveal that the time of extraction, besides affecting the yield and chemical structure of the K2-carrageenan, also significantly influence Mw. And consequently, the G’ and the ɳ was higher for the extracts with longer extraction times, comparing to the samples with similar conditions. 4.1.7 Natural carrageenans (HWE) vs less sulphated carrageenans (HAE) It is well-established in the literature that K2-carrageenans extracted with alkaline solvents are less sulphated, while those obtained through water extraction, reflect their natural, more sulphated state (Souza et al., 2023). Comparing natural (from the HWE) and less sulphated K2-carrageenans (from the HAE) across the three algae species revealed that the main distinction in chemical composition is that HAE extracts are consistently less sulphated than HWE extracts, as expected. The Mw of carrageenans within the same species was significantly higher (2–5 times) in HWE extracts compared to HAE extracts (see Table 8). This reduction in Mw, indicates that alkaline conditions promote carrageenan degradation, leading to lower molecular masses (Van De Velde et al., 2005; Souza et al., 2023). The broader PDI observed in some HAE extracts, highlights the degradation caused by alkaline conditions, which likely fragment carrageenan chains into molecules of varying lengths. This aligns with the expectation that alkaline treatments disrupt the structural integrity of natural carrageenans, leading to less uniform products (Van De Velde et al., 2005; Souza et al., 2023). The comparison between natural and less sulphated K2-carrageenans underscores the complementary roles of water and alkaline extractions in modulating carrageenan properties. Water extractions retain the native, more sulphated form with higher Mw, while alkaline extractions favour desulphation and lower Mw. These findings suggest that strategic use of SE and extraction conditions can optimize the carrageenan recovery and tailor its properties for specific applications, offering valuable 67 insights into the biorefinery potential of red algae. For example, food-grade carrageenan typically has a molecular mass greater than 100 kDa, with commercial products ranging from 200–800 kDa (Torres, Flórez-Fernández, et al., 2019). So, from the same algae at different extraction times and with different solvents, different carrageenans can be obtained to satisfy various types of products. 4.1.8 Texturizing behaviour The texturizing capacity of the carrageenan is a key factor in their commercial application and value. Quantitative results regarding the texturizing behaviour (gel elasticity, viscosity and gelling temperature) can be observed in Table 8. Table 10 shows that when 1 M NaCl was added to a 2% (w/v) carrageenan solution, all the samples of 1 h and 30 min hot water SE gelled after 24 h at 25 ºC, in all species, whereas the 10 min hot water SE extraction samples remained liquid. In comparison, the control samples did not form gels, except for the 1 h hot water control from C. crispus , which exhibited a similar elastic modulus G0 to its respective sequential extract (as showed in Table 8). So, overall, the “natural” K2-carrageenans obtained from the sequential HWE had better elasticity than the corresponding controls, suggesting that the sequential extraction process contributed to improve the gel quality. This is probably due to the lower amount in precursors for the SE extracts, as many of the carrageenan samples imaged in Table 10 show similar Mw but different gelling behaviour. Results recently reported for a set of hybrid carrageenans extracted from commercial seaweeds with hot water, showed that when the hybrid carrageenan composition is close to a balanced amount in Iand K-carrageenans, no gel can be formed with 1 wt.% in 1 M NaCl (Moraes & Hilliou, 2024). This observation aligns with some of the non-gelling behaviour of the control samples, as suggested by the chemical characteristics presented in Table 8. If a threshold of at least 55 mol% of either Ior Kcarrageenan is here hypothesized for gel formation in NaCl, the lack of gelation in the controls can be attributed to their composition, independently of the Mw effects. But it cannot explain the formation of gels in HWE SE samples, when they show a very close carrageenan composition and molecular masses to the respective controls. In contrast, all HAE samples (both SE and control) remained liquid. While the HWE samples, with higher sulphate content (Table 8), were expected to exhibit weaker gels or lower viscosities due to the presence of carrageenan precursors incapable of gelling, the larger molecular mass of water-extracted K2-carrageenans likely contributed to gel formation. This highlights the significance of molecular mass in gel setting, as smaller polysaccharides from the alkaline extracts may struggle to aggregate or form 68 helices. Considering the above discussion for the HWE, the gel setting of hybrid carrageenans in NaCl depends on a complex interplay between the chemical structure, the length of κ and ι blocks and the molecular mass. As such, the gelling properties of these carrageenans (from the HWE and HAE) are better studied in the presence of KCl (Moraes & Hilliou, 2024) or even in mixtures of salts that favour the gelling of the K-carrageenan blocks (Van de Velde et al., 2005). Nevertheless, gelling in the presence of a single cation (Na⁺, from NaCl and from the alkali chosen in extraction) was preferred here, in part to highlight the role of ι blocks in the hybrid carrageenan structure. Table 10. Comparison of gels/liquids of solutions of carrageenan (2%(w/v) car + 1M NaCl), after cooling for 24 h at 25ºC, from Mastocarpus stellatus, Chondrus crispus, and Gigartina pistillata , after different extraction times (1h, 30min, 10min) using HWE, at 90ºC. The first row shows results from the sequential extraction (SE), while the second row displays the controls Gels The gelling temperature (Tg) is reported here as the temperature for the gel point, where the formation of helices and junction zones upon cooling leads to the development of a three-dimensional elastic network. For C. Crispus , for both the control and sequential 1 h hot water extracts, the graphs did not show a clear crossover point where G’ equals G’’, being the G’ always higher than the G’’, in the study temperature range (25 ºC – 85 ºC). In other words, the samples are never truly liquid, making it impossible to determine Tg accurately. So, it is assumed that Tg is higher than 85 ºC (see Table 8), see Appendix H for more detailed information about the rheological behaviour of the samples. Algae M. stellatus C. crispus G. pistillata Time 1h 30min 1h 30min 10min 1h 30min 10min SE Control 69 When extraction time doubled from 30 min to 1 h, the Tg for M. stellatus decreased, this coincided with the presence of precursors (more than 30 mol %) in the 1 h K2-carrageenan extract, whereas no precursors were detected in the 30 min extract. In contrast, for G. pistillata and C. crispus , Tg increased with extended extraction time, even though the 1 h K2-carrageenan extracts also exhibited higher precursor contents, similar to M. stellatus . Prior research suggests that for K2-carrageenans containing only κand ι-carrageenan, Tg remains independent of carrageenan composition (van de Velde, 2005). Similarly, studies by Hilliou et al. (2006) and Azevedo et al. (2013, 2015) found no correlation between Tg and the chemical structures of hybrid carrageenans from M. stellatus , C. crispus , and Ahnfeltiopsis devoniensis , which displayed compositions similar to those shown in Table 8. Also, Hilliou et al., (2006) concluded that the time of extraction (30 min – 6 h) didn’t seem to affect the Tg. Thus, the lack of a clear relationship between Tg and chemical structure here is consistent with previous findings. Even so, the measured Tg listed in Table 8, ranging from 26 - 39°C, are similar to the Tg reported in the literature for similar gelling conditions (Souza et al., 2011) and such values are suited to a wide range of food and non-food applications (Torres et al., 2018). Also, K2-carrageenans with Tg above 85 ºC can find applications in various industries due to their stability and functional properties under heat (Chen et al., 2022). Among the samples that formed gels, the 1 h hot water SE extract of M. stellatus produced the most elastic K2-carrageenan, with a G0 of 376,32 Pa, while the 30 min hot water SE extract of G. pistillata yielded the least elastic gel, with a G0 of 30,28 Pa. These results highlight the impact of extraction conditions and algae on the structural and rheological properties of carrageenan, particularly its gel elasticity. In M. stellatus and G. pistillata , increasing the extraction time from 30 min to 1 h significantly improved gel elasticity, with G0 increasing approximately 2,43-fold. However, in C. crispus , the opposite trend was observed: the 30 min SE extract exhibited higher G0 than the 1 h extract, with the latter showing a reduction of more than half in gel elasticity. This divergence is consistent with prior studies, such as those by Van de Velde et al. (2005), which demonstrate a direct correlation between κ-carrageenan content and gel elasticity. The 30 min SE extract of C. crispus contained fewer precursors (2,73 mol % mu-carrageenan) and a higher κ content (68,66 mol %) than the 1 h extracts (SE and control), which showed higher precursor levels (7,66–10,78 mol %) and lower κ content (approximately 60 mol %) (show in Table 8). This chemical composition explains the superior elasticity of the shorter extraction time sample, as the increased precursor content in the 1 h extract likely hindered the formation of robust helices. The same G0 enhancement with the larger content in κ-carrageenan is found within gelling 70 extracts from M. stellatus and G. pistillata. To note that although the 1h SE water extract from G. pistillata has a higher κ content and Mw compared to the same extract from M. stellatus , the PDI of the latter is more than four times higher, suggesting that the higher G0 observed in M. stellatus could be attributed to its broader Mw distribution, indicating the presence of carrageenan with longer chains. As seen before the rheological properties of K2-carrageenan gels vary significantly between algae and extraction conditions, these differences directly influence their potential applications. The highly elastic gel from M. stellatus (376,2 Pa) is ideal for firm food products like jellies and puddings, as well as durable non-food uses such as wound dressings and cosmetic hydrogels. In contrast, the softer gel from G. pistillata (30,28 Pa) suits spreads, drinkable gels, and flexible films. Also, the gel's yield strain, when G’=G’’ (see Table 8) reflects its resistance to deformation before fluidization and is a key parameter for tuning texture and performance in various applications (Franck, n.d.). So, by optimizing extraction conditions, these gels can be tailored to meet specific industrial needs (Mugdha Bhat et al., 2020; Russo Spena et al., 2024). Liquids To further evaluate the rheological behaviour of the liquid carrageenan samples, viscosity (ɳ) as a function of shear rate (ɣ) was analysed. This included both the ramp-up (0 to 1000 s⁻¹) and ramp-down (1000 to 0 s⁻¹) in shear rates, allowing for evidencing any flow hysteresis (see Table 11). The C. crispus , doesn’t have any data for the alkaline samples, since there wasn’t enough mass extracted to do the tests, as said before. As for the 10 minutes hot alkaline SE from the G. pistillata the rheometer was unable to detect the viscosity, as it was close to the instrument's sensitivity limit and similar to the viscosity of water. For comparative purpose, the viscosity values at a shear rate of 500 s⁻¹ are reported in Table 8, since the solutions show no flow hysteresis when the shear rate is higher. Additionally, the elastic moduli (G') at 1 Hz and 25ºC, G1, was extracted providing insight into their structural rigidity, and as expected the G’ of the liquid samples are very low and close to the sensitivity limit of the rheometer, which may not fully represent the true elastic moduli. The rheological plots are given in Appendix H. Analysing the viscosity ramps for hot water extracts, distinct behaviours were observed among the K2-carrageenan solutions from different species. For M. stellatus , both samples exhibited nonNewtonian behaviour, and clear hysteresis was observed. This hysteresis indicates that, under low shear rates, the viscosity did not recover its initial value within the experimental time frame, suggesting 71 structural changes under shear stress. These changes likely result from the breakdown or rearrangement of carrageenan aggregates at higher shear rates, a phenomenon consistent with the polymer’s high molecular mass and aggregation propensity. Conversely, for the same extraction (HWE), C. crispus K2-carrageenan solutions, while also nonNewtonian, exhibited limited hysteresis. For the 30 min control sample, the behaviour suggests less pronounced aggregation or smaller aggregate sizes that remain relatively unaffected within the applied shear rate range. This is likely due to the smaller molecular mass, or fewer aggregate-forming interactions compared to M. stellatus and G. pistillata. Indeed, for the G. pistillata , hysteresis was prominent in all HWE samples. The 30 min control sample exhibited a significant mismatch between flow curves near 0,01 s⁻¹ shear rate, indicating more fragile carrageenan aggregates or a higher degree of breakdown under shear stress, limiting viscosity recovery. Interestingly, the 10 min control extract showed a viscosity approaching that of water (~1 mPa·s) (see Table 8), behaving as a Newtonian fluid at shear rates above 1 s⁻¹. At lower shear rates, the rheometer’s torque sensitivity limited the measurements, resulting in curves with slopes approaching -1, for the previous sample. This behaviour highlights the influence of molecular structure and aggregation state on the observed viscosity profiles. Additionally, the hysteresis (Table 11) observed in the M. stellatus and G. pistillata extracts suggests that their carrageenans had a higher tendency to initially aggregate compared to C. crispus , that likely indicates that the higher molecular mass favours loose aggregation between chains, which are breaking down under shear stress, and need more time than the experimental time to re-aggregate at smaller shear rates. For the HAE, M. stellatus sequential K2-carrageenan extracts and control samples also exhibited hysteresis. And, despite having similar molecular masses, differences in viscosity could be attributed to compositional variations, such as precursor content. In G. pistillata , the 30 min and 10 min SE extract behaved essentially as a Newtonian fluid , whereas data scattering at lower shear rates impedes a clear conclusion about flow hysteresis or limit of torque sensitivity. Overall, when comparing liquid samples (Table 8), sequential extracts generally exhibited higher or comparable viscosities at 500 s⁻¹ compared to controls. There is an exception for M. stellatus , the 1 h control extract showed slightly higher viscosity than its sequential counterpart, possibly due to a marginally higher κ-carrageenan molar content (~ 4% higher). Longer extraction times across all samples were associated with higher viscosities, attributable to the recovery of higher molecular mass carrageenans. Shorter extraction times yielded carrageenans with shorter chain lengths, which dissolved more readily but formed weaker structures. This aligns with studies showing that higher molecular mass 78 The results revealed notable alterations in the chemical composition, more pronounced in samples B-87 and Gigartina , where the appearance of a prominent absorption band around 1110 cm-1 was observed, after ultrasound. This band corresponds to O-H deformation, C-O stretching, and ring vibrations of polysaccharides, such as carrageenans (Masaki et al., 2017), that may result from the breakup of the glycosidic bonds. However, the modification in the 1110 cm-1 band was not observed in K2 with higher κ-carrageenan content. Additionally, evidence of desulphation was detected aligning with findings from the referenced study (Tecson et al., 2021). So, the structural modifications caused by the mechanical breakdown of the Mw encompasses the molecular structure of ultrasonicated hybrid carrageenans and thus impact their gelling properties (Souza et al., 2011). The stability and gelation performance of ultrasonicated hybrid carrageenans, therefore, appear to depend on both energy input and the different starting macromolecular chemical composition, as there is a distinctive sulphate composition provided by the κ and ι units in the K2-carrageenans, being that the κ-carrageenan has only one sulphate group and the ι-carrageenan has two sulphate groups (Souza et al., 2023). Although, the distinction in numbers of sulphate groups alone does not provide conclusive evidence for significant differences in their resistance to mechanical breakdown. Further chemical analyses are recommended to confirm these findings and provide deeper insight into the structural changes at each time point. 4.2.3 The impact of the Mw on the Rheology Some carrageenans required no more than 2 h of ultrasonication to lose their gelling capability at 1 wt.% in 0,1 M KCl, as shown in Table 13, while others needed over 16 h to reach that state. This was expected, given their different initial chemical compositions and Mw. The table lacks some data due to some reasons: the B-87 samples were the only ones measured in a rheometer where the control of the gel thickness was different and didn’t allow to extract Ton; for the Gigartina sample at 1 h, it was not possible to determine Ton, as the solution continued to increase in thickness throughout the tested temperature range (25 ºC–80 ºC). The corresponding rheological graphs from the different samples of each algae can be found in Appendix J 79 Table 13. Summary of κ-content (mol%), molecular mass (Mw), polydispersity index (PDI), phase behaviour postgel test for (0,1% (w/v) carrageenan + 0,1 M KCl), elastic modulus G' (at 1 Hz, 25 ºC, under 0,1% or 50,3% strain for gels (G0) and liquids (G1), respectively), gelation temperature (Tg), onset temperature (Ton) of the gels, and viscosity (ɳ) at high shear strain for the liquids samples of the different K2-carrageenans ultrasonicated at various times. With “nd” representing no data and grey lines representing the gel samples. For each carrageenan and time point, values of Mw and PDI with different letters indicate statistically significant differences (p < 0,05) 4.2.4 Storage Modulus, G’ The data in Table 13 reveals a clear correlation between molecular mass and the storage modulus (G’), representing the elasticity of each sample: as the Mw reduces, the lower the G’. The molecular mass dependence of G’ is qualitatively in concordance with the single literature which concluded that the elasticity of samples of 1 wt.% K-carrageenan in 0,1 M KCl increases with larger Mw (Rochas et al., 1990). Such correlation is less evident for sample B-87 since the ultrasonication did not produce carrageenans with very different molecular masses. These results (in Table 13) contradict the conclusions of the study of Souza et al. (2011) performed with hybrid carrageenans dissolved in a different salt, 0,1 M NaCl. Under such gelling conditions, the highest elasticity is obtained with lowest molecular mass. However, G’ is found to decrease with the increase of the ι-content in hybrid carrageenans, having similar Mw (Souza et al., 2011). Here, Algae Time (h) 0Gel 20,76 >85 nd - 1 2,64 ± 0,46 a3,26 ± 0,09 aGel 25,05 >85 nd - 2 3,02 ± 0,10 a2,72 ± 0,06 aLiquid 0,0322 - - 10,09 3 2,52 ± 0,01 a2,51 ± 0,00 aLiquid 0,0466 - - 9,65 4 2,67 ± 0,10 a2,32 ± 0,06 aLiquid 0,856 - - 12,06 0 15,40 ± 0,81 a2,70 ± 0,16 aGel 638,19 60,03 53,27 - 1 5,57 ± 0,67 b2,12 ± 0,00 aGel 112,96 55 nd - 2 5,79 ± 0,04 b2,00 ± 0,05 aLiquid 1,64 - - 26,97 3 5,07 ± 0,03 b,c 1,96 ± 0,03 aLiquid 0,976 - - 24,37 4 3,29 ± 0,01 c1,97 ± 0,01 aLiquid 0,655 - - 17,03 06,49 ±0,06 a2,83 ± 0,03 aGel 3518,53 57,68 55 - 6 2,42 ± 0,01 c2,11 ± 0,07 aGel 4232,02 62,07 56,56 - 12 1,83 ± 0,01 d2,11 ± 0,04 aGel 3542,98 65,61 52,96 - 16 3,12 ± 0,03 b2,17 ± 0,06 aGel 2189,33 62,92 51,07 - 20 1,42 ± 0,01 e2,04 ± 0,02 aLiquid 4,85 - - 32,79 0 6,66 ± 0,17 a2,51 ± 0,01 aGel 4235,96 62,76 54,68 - 6 1,87 ± 0,06 c1,99 ± 0,01 aGel 809,77 58,36 43,7 - 12 1,53 ± 0,02 d1,97 ± 0,00 aGel 4460,8 68,96 56,56 - 14 2,37 ± 0,04 b2,00 ± 0,01 aGel 1188,12 66,78 50,91 - 16 1,08 ± 0,00 e2,01 ± 0,03 aLiquid 0,00466 - - 7,08 0 9,64 ± 0,26 a3,41 ± 0,00 aGel 5543,03 63,33 54,52 - 6 2,59 ± 0,03 b2,03 ± 0,01 aGel 3603,74 67,52 54,84 - 12 1,61 ± 0,02 c2,01 ± 0,01 aLiquid 1,38 - - 14,16 16 0,83 ± 0,01 d1,92 ± 0,03 aLiquid 0,401 - - 5,4 20 0,77 ± 0,01 d1,91 ± 0,03 aLiquid 0,55 - - 10,51 nd nd B-87 Hybrid Carrageenans κ (mol %) 4 ± 2,5 PDI Tg (ºC) TON (ºC) ɳ (mPa/s) at 500 s¯¹ Phase ( 1%(w/v) car + 0,1 M KCl) G0/G1(Pa) Mastocarpus Gigartina BAT 18-09 C-25 Mw (g/mol) · 10⁵ 42 ± 1,8 69 ± 0,4 56 ± 2,9 90 ± 4,5 80 only two samples offer such a comparison but in KCl since Mastocarpus and BAT 18-09 show similar initial Mw. However, the hybrid carrageenan chemical composition does not show sufficient difference in the ι-content to draw a clear conclusion. The 6h BAT 18-09 sample appears to be an outlier, in terms of the G0, as although the Mw decreased, as expected, its G' value is notably lower, compared to other samples from the same alga with similar Mw. This suggests a possible experimental error, during the rheology phase, potentially related to the addition of the salt (0,1 M KCl). Given the fact that only a very small amount of KCl (0,0373 g) was required, there may have been inaccuracies in weighing, which could have affected the sample's rheological properties. Repeating this sample measurement would be necessary to verify if the result was an anomaly due to sample preparation or an actual material property change. The differences in the behaviour of K2-carrageenan, from different algae, when exposed to ultrasonication may also be attributed to variations in helix aggregation within their carrageenan structures, where ι-helices might exhibit greater resistance to mechanical stress compared to κ-helices. This difference aligns with observations of I gels displaying more curved helix structures, in contrast to the rod-like helices and more complex, branched aggregation patterns seen in K gels (Hilliou, 2021). The single-helix mesh in I gels results in softer textures, whereas the super helical network in K gels, especially in the presence of K⁺, produces firmer gels (Souza et al., 2023; Stephen et al., 2006). For K2carrageenans, early studies suggest a structure resembling with K-carrageenan, but with additional branching and unique aggregation behaviour that complicates understanding its mechanical properties (Souza et al., 2023). But there is still a long way to go until a complete understanding of the interaction between such macromolecular structures and the properties of the K2 gel, so the discussion cannot go further from the helix perspective. 4.2.5 Critical Molecular Mass, Mc According to Rochas et al. 1990, the storage modulus (G’) increases steadily with Mw until reaching a critical value, Mc, where it plateaus for a given salt concentration. For pure K-carrageenan the Mc given was 1,8·105 g/mol. However, in this work it was not possible to obtain clear constant G’ values for gels, making it difficult to find a Mc, as shown in Figure 17. However, Rochas et al. 1990 extrapolated from their rheological data on gels that for Mw below 0,4·105 g/mol, no K-carrageenan gels can be formed for 1 wt.% carrageenan in 0,1 M KCl. This critical mass is much below the range of Mw (between 1,6·105 g/mol and 2,6·105 g/mol, see Table 13 and Figure 17) that separates liquids from gels with sample C- 81 25 (the κ-richer K2-carrageenan). This suggests that the extrapolation proposed is not valid, or that the drop in the sulphate content in C-25 that accompanies the decrease in Mw impedes a fair conclusion on such critical mass. Figure 17. Storage modulus, G’, as function of the molecular mass, Mw, of the different products before and after ultrasound (1 % (w/v) K2-carragenan + 0,1 M KCl) of the five different algae. The hybrid carrageenans with the initial higher molar content of κ-carrageenan, Mastocarpus , BAT 18-09 and C-25, had similar initial Mw and kind of similar behaviour, so it was possible to combine them in a graph, in the orange square. The gel samples are represented by the full symbols and the liquid sample as the empty symbols. There is a possible secondary threshold Mc2, where (a very low) Mw stabilizes with further ultrasonication, showing little additional change over time, as seen in Figure 15, and discussed at the beginning of section 4.2.1, it seems that κ-rich samples (like Mastocarpus and C-25) had a Mc2 close to 1·10⁵ g/mol, while more ι-rich hybrids, such as Gigartina and B-87, may have Mc2 close to 3·105 g/mol. The existence of an ultrasound resistant Mc2 may be possible, since the bonds that remain to be broken in carrageenans are very stable (e.g., C-C). Furthermore, more information is required to understand the rheological meaning of the value of Mc and its possible relation to the mesh size of the gel. For κ-rich K2-carrageenans, the minimum Mw required for gel formation (hereafter referred to as Mwgel) at the study conditions, 1% wt. of K2carrageenan with 0,1 M KCl, appears to be very close to the critical molecular mass, Mc2. In contrast, B-87 K2-carrageenan, which is predominantly ιRich in κ mol.% 82 carrageenan (96 mol%), displays weak gelling properties from the outset. For this type, the liquid samples exhibit G’ values that are relatively insensitive to Mw, likely because their Mw values (3,02–2,52·105 g/mol), which result in minimal elasticity, are near Mc2 (3·105 g/mol). However, a sample of B-87 forms a gel at Mw 2,64·105 g/mol, and when examining the PDI it is evident that liquid samples (PDI ≈ 2) have a narrower molecular size distribution compared to the gel-forming B-87 sample (PDI = 3,3), even though statically they are similar. This suggests that, even when the average Mw is near Mwgel, only a fraction of molecules with sufficiently high Mw may contribute to gelation. For K2-carrageenan derived from Gigartina , the transition from gel to liquid phase occurs at a Mw breakpoint near 5,5·105 g/mol (Mwgel), which is above Mc2 (3·105 g/mol). This discrepancy may stem from the presence of precursor molecules, specifically 19 ± 5 mol% nu-carrageenan. While nu-carrageenan does not directly affect Mc2, it may influence the rigidity of the polymer chains, thereby reducing the gelation ability of the sample. 4.2.6 Thermal behaviour of the gels The Tg of Gigartina carrageenan samples decreases with a reduction in Mw, as seen in Table 13. However, for the more κ-rich K2-carrageenans (from Mastocarpus , BAT 18-09, and C-25), Tg tends to increase as Mw decreases, with BAT 18-09 6h standing out as an outlier in this group, as discussed before. A study by Van de Velde et al. (2005) on K2-carrageenans from Mastocarpus , with various chemical structures, reported that gelling temperature is independent of biopolymer chemistry in polysaccharides with more than 50 mol% κ-carrageenan disaccharide units. This suggests that Tg dependency in these carrageenans may be more influenced by Mw. Additionally, the gradual Tg variation of approximately 2 - 10 ºC likely indicates shifts in the helical structure. Ultrasonication time is also expected to affect TON by reducing Mw over time, yet the relationship between ultrasonication duration and TON appears inconsistent across samples. This indicates that the thermal properties of carrageenans may be sensitive to distribution and density of the polymer network rather than merely the average Mw value. 4.2.7 Viscosity of the liquids The viscosity of the liquid solutions after varying durations of ultrasonication is shown in Table 13. In general, as molecular mass declines, viscosity also decreases due to the smaller size and lower sulphation (confirmed by the FTIR) of the K2-carrageenan molecules, which reduces resistance to flow, as the proportion of sulphate fractions and the equilibrium of cations in the water solution determine the 83 viscosity of solutions or strength of gels formed by carrageenans (Campos et al, 2009). For B-87, since the Mw remains relatively constant (close to 3·10⁵ g/mol), viscosity also stays approximately the same. The C-25 16h sample presents an unexpected result: its viscosity is less than half of the 20h sample, despite having a similar Mw and comparable elasticity (G1) (as seen in Table 13). This discrepancy could be attributed to minor inconsistencies during sample preparation, such as slight variations in sample hydration, concentration, or solution heterogeneity. Additionally, this testing was done in the summer season, and the higher temperature of the cooling system may have lowered the viscosity. Repeating viscosity measurements and doing a chemical structure analysis for this sample could help determine whether this observation is due to experimental variability or a unique characteristic of the K2-carrageenan at this ultrasonication stage. 84 5. CONCLUSIONS AND FUTURE WORKS In conclusion, the sequential extraction (SE) procedure and subsequent ultrasonication experiments collectively advance the understanding of optimizing K2-carrageenan extraction and molecular tailoring from carrageenophytes. In some algae, SE demonstrated yields of K2-carrageenans comparable to those obtained via conventional extraction methods with longer processing times, further supporting its industrial relevance. The SE protocol proved highly effective in fractionating phycobiliproteins, chlorophyll-a, carotenoids, and carrageenans (natural and less sulphated) into distinct fractions, showcasing its potential for sustainable industrial applications. Its design aligns with biorefinery principles, promoting streamlined and efficient processes. While chlorophyll-a and carotenoids are co-extracted in the same solution, implementing advanced separation techniques like High-Performance Liquid Chromatography (HPLC) could provide deeper insights into their specific composition and concentrations. When comparing the efficiency and quality of SE to the control, of the carrageenans extracted, SE consistently facilitates the recovery of high molecular mass (Mw) K2-carrageenans during hot water extraction (HWE), particularly from M. stellatus and G. pistillata. Moreover, overall, SE extracts demonstrate superior rheological properties under the studied conditions, including enhanced gel elasticity and comparable or higher viscosities in liquid states. This is attributed to the pre-treatments, such as cold-water extraction (CWE), which yield less sulphated carrageenans in subsequent steps (in most cases), thereby improving gelling properties. Besides, K2-carrageenans extracted under alkaline conditions (HAE) are less sulphated but exhibit significantly lower Mw and lower rheological properties, compared to those obtained via HWE. Ultrasonication effectively reduces molecular mass, a process strongly influenced by the chemical composition of K2-carrageenans, further elucidating the relationship between Mw and the elastic properties of gels. The reduction in Mw correlates with a decrease in gel elasticity, stabilizing near Mc2 (~1·10⁵ g/mol for κ-rich and ~3·10⁵ g/mol for ι-rich hybrids), where further degradation has minimal impact. Although a critical molecular mass (Mc) for gelation was not clearly defined, degraded K2carrageenans exhibit reduced viscosity and gel elasticity, underscoring the essential role of molecular mass in determining functional properties. This interplay between chemical composition and Mw highlights the complexity of optimizing extraction parameters to enhance both efficiency and product functionality. Extended extraction times typically result in higher molecular mass carrageenans with enhanced gel elasticity, though they also lead to higher sulphation levels, an apparent contradiction. Interestingly, C. crispus displayed an inverse 85 behaviour in the HWE. Additionally, shorter extraction times occasionally produced comparable yields, particularly in pigment extraction, underscoring the necessity for species-specific optimization. Achieving a balance between energy efficiency and extraction outcomes is essential. The findings from the SE process and the ultrasonication study complement each other, offering a comprehensive understanding of how to optimize K2-carrageenan extraction and tailor its properties. This work underscores the critical influence of extraction sequence, duration, and solvent selection on product quality. By addressing key gaps in the literature, such as the impact of extraction pre-treatments on carrageenan sulphation and the relationship between Mw and gel functionality, it lays the groundwork for future innovations in carrageenophytes valorisation. Additionally, it contributes to the development of sustainable and cost-effective biorefinery methods, enhancing the valorisation of red seaweed through the extractive-extrusion approach. Future Research For future research, evaluating the gel strength of K2-carrageenan extracts in the presence of different cations (e.g., KCl) is also suggested to enhance their practical applications in various industries. Investigating the sequential extraction protocol's order, varying extraction times, and temperature ranges is also recommended to optimize yield and product quality. Additionally, comprehensive analyses of phenolic compounds and soluble proteins within the extracts should be performed, accompanied by bioactivity scans Assessing residual fractions, particularly the waste fraction, through weighing and compositional analysis could reveal new opportunities for valorisation, including the identification of bioactive compounds that support a holistic utilization of biomass resources. Furthermore, studies on alkaline extracts of C. crispus should be completed, particularly in areas where current data, limited by insufficient mass, does not allow for clear conclusions. In alignment with the project under which this work is framed, future efforts are foreseen to adapt the sequential extraction protocol for use in an extruder. This adaptation could streamline the process, enhancing scalability and efficiency while maintaining eco-friendly practices. To achieve this, it will be necessary to investigate the operational parameters of extrusion and their impact on product quality and yield, refining the protocol to ensure its applicability to industrial processes. For molecular mass studies focusing on the elastic properties of K2-carrageenan, it is essential to include pH monitoring throughout the process and perform chemical analyses on all samples to improve the accuracy of degradation assessments. Exploring additional conditions, such as varying the 86 types of salts and carrageenan concentrations, may provide a more comprehensive understanding of their effects on gel elasticity (G') and other rheological behaviours. Furthermore, evaluating additional time points during ultrasonication is recommended to better capture the progression of molecular degradation and its impact on functional properties. This approach would allow for a more detailed understanding of the degradation process. 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N., Al-Ghaili, H., & Benz, R. (2016). A review on the valorization of macroalgal wastes for biomethane production. Marine Drugs , 14 (6). https://doi.org/10.3390/md14060120 94 APPENDIX Appendix A – Absorbance of the liquid extracts from the CWE Calculation example of yield of the R-PE of the different extracts: Sample: Test 1 of 60 min – Mastocarpus stellatus R-PE (mg/mL) = 0,1247· [(A564–A730) – 0,4583 · (A618– A730)] (1) R-PE = 0,1247· [(0,0940–0,0170) – 0,4583 · (0,0460– 0,0170)] = 0,0079 mg/mL (0,0079 mg/mL) / (2,004 g/60 mL) = 0,2365 mg/g of R-PE. Table A.1. Absorbance, yield and Standard Deviation (SD) of the R-PE extract from the CWE for the sequential extraction (60, 30 and 10 minutes) of algae M. stellatus Table A.2. Absorbance, yield and Standard Deviation (SD) of the R-PE extract from the CWE for the sequential extraction (60, 30 and 10 minutes) of algae C. Crispus 1 2 3 1 2 3 1 2 3 2,004 2,007 2,0015 2,0076 2,0083 2,001 2,0322 2,0168 2,0175 0,0940 0,1240 0,1340 0,2250 0,1840 0,1950 0,2020 0,2470 0,2100 0,0460 0,0720 0,0740 0,1530 0,1140 0,1250 0,1370 0,1710 0,1410 0,0170 0,0310 0,0320 0,0920 0,0610 0,0700 0,0830 0,1030 0,0840 0,0079 0,0093 0,0103 0,0131 0,0123 0,0124 0,0118 0,0141 0,0125 0,2379 0,2767 0,3093 0,3915 0,3677 0,3731 0,3470 0,4186 0,3704 Cold Water Extraction (16h) 0,3436 Mastocarpus stellatus 60 min 30 min 10 min R-PE (mg/mL) R-PE (mg/g) Average R-PE (mg/g) SD R-PE (mg/g) Algae Time Test Initial dry algae mass (g) Abs 564 Abs 618 Abs 730 0,0580 1 2 3 1 2 3 1 2 3 2,0101 2,018 2,0267 2,0076 2,0083 2,001 2,0052 2,0044 2,0166 0,0870 0,1540 0,0940 0,2290 0,2120 0,2210 0,1150 0,2170 0,1780 0,0670 0,1200 0,0720 0,1840 0,1700 0,1780 0,0880 0,1730 0,1410 0,0440 0,0800 0,0480 0,1270 0,1190 0,1270 0,0570 0,1200 0,0980 0,0040 0,0069 0,0044 0,0095 0,0087 0,0088 0,0055 0,0091 0,0075 0,1208 0,2064 0,1292 0,2828 0,2594 0,2641 0,1634 0,2714 0,2237 Cold Water Extraction (16h) 0,2135 0,0624 Chondrus crispus 60 min 30 min 10 min R-PE (mg/mL) R-PE (mg/g) Average R-PE (mg/g) SD R-PE (mg/g) Algae Time Test Initial dry algae mass (g) Abs 564 Abs 618 Abs 730 95 Table A.3. Absorbance, yield and Standard Deviation (SD) of the R-PE extract from the CWE for the sequential extraction (60, 30 and 10 minutes) of algae G. pistillata Appendix B - Absorbance of the liquid extracts from the EE For an example of the calculation see Appendix A, for more information. 1.Sequential Extraction Table B.1.1. Absorbance, yield and Standard Deviation (SD) of the Chlorophyll-a and Carotenoid extract from the EE for the sequential extraction (60, 30 and 10 minutes) of algae M. stellatus 1 2 3 1 2 3 1 2 3 2,0043 2,031 2,0618 2,0157 2,0045 2,0031 2,0019 2,0115 2,0123 0,1400 0,1420 0,1470 0,1680 0,1550 0,2000 0,1690 0,2050 0,2030 0,0900 0,0920 0,0940 0,1120 0,0990 0,1260 0,1030 0,1340 0,1360 0,0570 0,0590 0,0590 0,0710 0,0600 0,0770 0,0610 0,0820 0,0900 0,0085 0,0085 0,0090 0,0098 0,0096 0,0125 0,0111 0,0124 0,0115 0,2534 0,2500 0,2611 0,2903 0,2879 0,3756 0,3317 0,3689 0,3418 Cold Water Extraction (16h) 0,3067 0,0490 60 min 30 min 10 min Gigartina pistillata R-PE (mg/mL) R-PE (mg/g) Average R-PE (mg/g) SD R-PE (mg/g) Algae Time Test Initial dry algae mass (g) Abs 564 Abs 618 Abs 730 1 2 3 1 2 3 1 2 3 2,004 2,007 2,0015 2,0076 2,0083 2,001 2,0322 2,0168 2,0175 0,056 0,068 0,086 0,066 0,103 0,081 0,084 0,073 0,069 0,027 0,031 0,042 0,031 0,054 0,041 0,036 0,033 0,031 0,06 0,067 0,086 0,063 0,094 0,076 0,083 0,073 0,069 0,111 0,117 0,148 0,104 0,15 0,124 0,138 0,13 0,121 0,002 0,003 0,007 0,006 0,017 0,014 0,001 0,002 0,002 1,3383 1,3723 1,6971 1,1639 1,5720 1,3226 1,6121 1,5477 1,4320 40,0692 41,0245 50,8748 34,7860 46,9641 39,6582 47,5971 46,0452 42,5861 0,216 0,26 0,316 0,24 0,344 0,268 0,332 0,284 0,268 6,4671 7,7728 9,4729 7,1727 10,2773 8,0360 9,8022 8,4490 7,9703 6,1294 8,4954 1,6025 5,9820 7,9043 1,5072 Time Test Initial dry algae mass (g) 43,9895 40,4694 Mastocarpus stellatus 60 min 30 min 10 min Chloro-a (μg /mL) Algae Ethanolic Extraction 95% (v/v) Abs 480 Abs 632 Abs 652 Abs 665 Abs 750 2,5653 0,9501 45,4095 8,7405 Chloro-a (μg /g) Average Chloro-a (μg /g) SD Chloro-a (μg /g) Carot (μg/mL) Carot (μg /g) Average Carot (μg /g) SD Carot (μg /g) 96 Table B.1.2. Absorbance, yield and Standard Deviation (SD) of the Chlorophyll-a and Carotenoid extract from the EE for the sequential extraction (60, 30 and 10 minutes) of algae C. crispus Table B.1.3. Absorbance, yield and Standard Deviation (SD) of the Chlorophyll-a and Carotenoid extract from the EE for the sequential extraction (60, 30 and 10 minutes) of algae G. pistillata 1 2 3 1 2 3 1 2 3 2,0101 2,018 2,0267 2,0076 2,0083 2,001 2,0052 2,0044 2,0166 Chondrus crispus 60 min 30 min 10 min Algae Time Test Initial dry algae mass (g) 0,029 0,028 0,025 0,047 0,027 0,029 0,035 0,032 0,032 0,021 0,021 0,016 0,03 0,018 0,02 0,026 0,025 0,024 0,043 0,045 0,035 0,057 0,038 0,042 0,048 0,046 0,043 0,084 0,094 0,072 0,111 0,075 0,082 0,091 0,087 0,08 0,003 0 0 0,001 0,002 0,002 0,007 0,007 0,008 1,0163 1,1881 0,9069 1,3546 0,9170 0,9999 1,0559 1,0063 0,9069 30,3355 35,3265 26,8496 40,4844 27,3949 29,9807 31,5960 30,1218 26,9840 0,104 0,112 0,1 0,184 0,1 0,108 0,112 0,1 0,096 3,1043 3,3300 2,9605 5,4991 2,9876 3,2384 3,3513 2,9934 2,8563 Chloro-a (μg /g) Average Chloro-a (μg /g) SD Chloro-a (μg /g) Carot (μg/mL) Carot (μg /g) Average Carot (μg /g) SD Carot (μg /g) Ethanolic Extraction 95% (v/v) Abs 480 Abs 632 Abs 652 Abs 665 Abs 750 Chloro-a (μg /mL) 29,5673 2,3555 3,0670 0,2556 30,8372 4,2607 3,1316 0,1863 32,6200 6,9324 3,9084 1,3833 1 2 3 1 2 3 1 2 3 2,0043 2,031 2,0618 2,0157 2,0045 2,0031 2,0019 2,0115 2,0123 60 min 30 min 10 min Gigartina pistillata Algae Time Test Initial dry algae mass (g) 0,158 0,108 0,185 0,12 0,083 0,072 0,098 0,12 0,064 0,108 0,074 0,131 0,087 0,061 0,06 0,064 0,079 0,047 0,251 0,174 0,295 0,199 0,139 0,135 0,148 0,185 0,11 0,491 0,346 0,579 0,405 0,286 0,282 0,288 0,359 0,219 0,004 0,002 0,003 0,003 0,003 0,008 0,003 0,003 0,004 6,0822 4,3169 7,1785 5,0773 3,5907 3,5199 3,5561 4,4370 2,7142 182,0757 127,5297 208,8995 151,1313 107,4782 105,4347 106,5810 132,3486 80,9270 0,616 0,424 0,728 0,468 0,32 0,256 0,38 0,468 0,24 18,4404 12,5258 21,1854 13,9306 9,5784 7,6681 11,3892 13,9597 7,1560 Chloro-a (μg /g) Average Chloro-a (μg /g) SD Chloro-a (μg /g) Carot (μg/mL) Carot (μg /g) Average Carot (μg /g) SD Carot (μg /g) Ethanolic Extraction 95% (v/v) Abs 480 Abs 632 Abs 652 Abs 665 Abs 750 Chloro-a (μg /mL) 106,6189 25,7108 10,8350 3,4356 172,8350 41,4645 17,3839 4,4254 121,3481 25,8133 10,3924 3,2096 97 2. Controls Table B.2.1. Absorbance, yield and Standard Deviation (SD) of the Chlorophyll-a and Carotenoid extract from the EE for the control extraction (60, 30 and 10 minutes) of algae M. stellatus Table B.2.2. Absorbance, yield and Standard Deviation (SD) of the Chlorophyll-a and Carotenoid extract from the EE for the control extraction (60, 30 and 10 minutes) of algae C. crispus 1 2 3 1 2 3 1 2 3 2,0685 2,0323 2,0160 1,9999 2,0026 2,0100 2,0062 2,0011 2,0030 0,0950 0,1020 0,0940 0,1280 0,1940 0,1020 0,0570 0,2510 0,1520 0,0620 0,0640 0,0660 0,1120 0,1640 0,0830 0,0410 0,2330 0,1370 0,1220 0,1390 0,1220 0,1420 0,2220 0,1220 0,0710 0,2670 0,1720 0,2090 0,2460 0,2030 0,1880 0,3110 0,1820 0,1150 0,3150 0,2240 0,0160 0,0060 0,0220 0,0870 0,1200 0,0530 0,0190 0,2020 0,1060 2,3429 2,9032 2,1906 1,2261 2,3421 1,5802 1,1729 1,3397 1,4155 67,9582 85,7118 65,1968 36,7841 70,1728 47,1713 35,0773 40,1688 42,4001 0,316 0,384 0,288 0,164 0,296 0,196 0,152 0,196 0,184 9,16606 11,3369 8,57143 4,92025 8,86847 5,85075 4,54591 5,87677 5,51173 Mastocarpus stellatus 60 min 30 min 10 min 9,6915 51,3760 17,0869 6,5465 2,0640 72,9556 11,1332 Algae Time Test Ethanolic Extraction 95% (v/v) Algae mass (g) Abs 480 Abs 632 Abs 652 Abs 665 Chloro-a (μg /mL) Chloro-a (μg /g) Average Chloro-a (μg /g) SD Chloro-a (μg /g) Carot (μg/mL) Carot (μg /g) Average Carot (μg /g) SD Carot (μg /g) 1,4557 39,2154 Abs 750 3,7533 5,3115 0,6877 1 2 3 1 2 3 1 2 3 2,0078 2,0014 2,0010 2,0060 2,0240 2,0277 2,0108 2,0131 2,0250 0,0400 0,0340 0,0440 0,0760 0,1040 0,0990 0,0470 0,0530 0,0940 0,0290 0,0280 0,0360 0,0720 0,1000 0,0950 0,0440 0,0480 0,0790 0,0470 0,0390 0,0480 0,0780 0,1060 0,1020 0,0510 0,0570 0,0990 0,0790 0,0580 0,0690 0,0900 0,1180 0,1160 0,0630 0,0720 0,1350 0,0120 0,0160 0,0240 0,0630 0,0910 0,0850 0,0350 0,0360 0,0550 0,8215 0,5038 0,5467 0,3183 0,3183 0,3680 0,3284 0,4180 0,9547 24,5497 15,1043 16,3933 9,5218 9,4372 10,8896 9,7981 12,4598 28,2887 0,112 0,072 0,08 0,052 0,052 0,056 0,048 0,068 0,156 3,34695 2,15849 2,3988 1,55533 1,5415 1,65705 1,43227 2,02672 4,62222 Abs 750 Algae Time Test Ethanolic Extraction 95% (v/v) Algae mass (g) Abs 480 Abs 632 Abs 652 Abs 665 Chloro-a (μg /mL) Chloro-a (μg /g) Average Chloro-a (μg /g) SD Chloro-a (μg /g) Carot (μg/mL) Carot (μg /g) Average Carot (μg /g) SD Carot (μg /g) Chondrus crispus 60 min 30 min 10 min 18,6824 9,9495 0,8152 1,5846 0,0631 5,1219 2,6347 0,6284 16,8489 9,9962 2,6937 1,6964 98 Table B.2.3. Absorbance, yield and Standard Deviation (SD) of the Chlorophyll-a and Carotenoid extract from the EE for the control extraction (60, 30 and 10 minutes) of algae G. pistillata Appendix C - Yield of the “natural” K2-carrageenan from the HWE (SE and Control) Calculation example of yield: Sample: Test 1 of 60 min – Mastocarpus stellatus 1.Sequential extraction Table C.1.1. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HWE for the sequential extraction (60, 30 and 10 minutes) of alga M. stellatus Note: Due to the insufficient mass, it was not possible further chemical testing and due the fact that it does not form a film, it is not possible to confirm that it is carrageenan after 10 minutes of extraction. 1 2 3 1 2 3 1 2 3 2,0510 2,0268 2,0000 2,0356 2,0421 2,0051 2,0038 2,0015 2,0039 0,2280 0,0960 0,1360 0,1660 0,1740 0,3870 0,1000 0,0970 0,0980 0,1160 0,0520 0,0720 0,1100 0,1110 0,2930 0,0510 0,0460 0,0450 0,2550 0,1130 0,1580 0,1780 0,1870 0,3960 0,1100 0,1080 0,1080 0,4480 0,1970 0,2720 0,2590 0,2790 0,5230 0,1900 0,1890 0,1910 0,0100 0,0080 0,0090 0,0570 0,0540 0,2080 0,0070 0,0010 0,0000 5,2833 2,2900 3,1624 2,3719 2,6599 3,6955 2,2042 2,2575 2,3004 154,5567 67,7921 94,8708 69,9113 78,1524 110,5825 66,0017 67,6746 68,8777 0,872 0,352 0,508 0,436 0,48 0,716 0,372 0,384 0,392 25,509508 10,4204 15,24 12,8512 14,1031 21,425365 11,1388 11,5114 11,7371 Abs 750 Algae Time Test Ethanolic Extraction 95% (v/v) Algae mass (g) Abs 480 Abs 632 Abs 652 Abs 665 Chloro-a (μg /mL) Chloro-a (μg /g) Average Chloro-a (μg /g) SD Chloro-a (μg /g) Carot (μg/mL) Carot (μg /g) Average Carot (μg /g) SD Carot (μg /g) Gigartina pistillata 60 min 30 min 10 min 86,2154 21,5010 16,1266 4,6314 67,5180 1,4444 11,4624 0,3021 105,7399 44,3917 17,0566 7,7069 1 2 3 1 2 3 1 2 3 2,004 2,007 2,0015 2,0076 2,0083 2,001 2,0322 2,0168 2,0175 Mastocarpus stellatus 60 min 30 min 10 min Algae Time Test Initial dry algae mass (g) 0,1059 0,1076 0,1092 0,0370 0,0507 0,0488 0,0093 0,0025 0,0035 5,284% 5,361% 5,456% 1,843% 2,525% 2,439% 0,458% 0,124% 0,173% 0,0051 0,0037 0,252% 0,180% Carrageenan (g) Average Carrageenan (g) SD Carrageenan (g) Yield (% dw) Average Yield (% dw) SD Yield (% dw) 0,371% Hot Water Extraction (90 ºC) 0,086% 0,0455 0,0074 2,269% 0,1076 0,0017 5,367% Y=(sample mass)/(algae mass) (4) Y= 0,1049/2,004 · 100 = 5,284 % d.w. 99 Table C.1.2. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HWE for the sequential extraction (60, 30 and 10 minutes) of alga C. crispus Table C.1.3. Yield and Standard Deviation (SD) K2-carrageenan extract from the HWE for the sequential extraction (60, 30 and 10 minutes) of alga G. pistillata 2.Controls Table C.2.1. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HWE for the control extraction (60, 30 and 10 minutes) of alga M. stellatus Table C.2.2. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HWE for the control extraction (60, 30 and 10 minutes) of alga C. Crispus 1 2 3 1 2 3 1 2 3 2,0101 2,018 2,0267 2,0076 2,0083 2,001 2,0052 2,0044 2,0166 Chondrus crispus 60 min 30 min 10 min Algae Time Test Initial dry algae mass (g) 0,6157 0,5230 0,7098 0,4674 0,5088 0,5903 0,2623 0,1927 0,2900 30,630% 25,917% 35,022% 23,282% 25,335% 29,500% 13,081% 9,614% 14,381% 26,039% Carrageenan (g) Average Carrageenan (g) SD Carrageenan (g) Yield (% dw) Average Yield (% dw) SD Yield (% dw) 30,523% Hot Water Extraction (90 ºC) 3,169% 0,2483 0,0501 12,358% 2,464% 0,6162 0,0934 0,5222 0,0625 4,554% 1 2 3 1 2 3 1 2 3 2,0043 2,031 2,0618 2,0157 2,0045 2,0031 2,0019 2,0115 2,0123 60 min 30 min 10 min Gigartina pistillata Algae Time Test Initial dry algae mass (g) 0,1253 0,2245 0,2400 0,1646 0,1389 0,1391 0,0854 0,0530 0,0732 6,252% 11,054% 11,640% 8,166% 6,929% 6,944% 4,266% 2,635% 3,638% 7,347% 3,513% 0,0622 0,0148 0,0164 9,649% Carrageenan (g) Average Carrageenan (g) SD Carrageenan (g) Yield (% dw) Average Yield (% dw) SD Yield (% dw) Hot Water Extraction (90 ºC) 0,1966 0,1475 0,0705 2,956% 0,710% 0,823% 1 2 3 1 2 3 1 2 3 2,0173 2,0159 2,0042 2,0098 2,0161 2,0046 2,0082 2,0037 2,0157 0,1976 0,2493 0,2301 0,1868 0,1897 0,2333 0,0000 0,0000 0,0000 9,795% 12,367% 11,481% 9,294% 9,409% 11,638% 0,000% 0,000% 0,000% Carrageenan (g) Average Carrageenan (g) SD Carrageenan (g) Yield (% dw) Average Yield (% dw) SD Yield (% dw) Hot Water Extraction (90 ºC) Algae mass (g) 0,0000 0,0000 0,2257 0,0261 11,214% 1,306% 0,000% 0,000% Algae Time Test 0,2033 0,0261 10,114% 1,321% Mastocarpus stellatus 60 min 30 min 10 min 1 2 3 1 2 3 1 2 3 2,0159 2,0134 2,0021 2,0148 2,0021 2,0039 2,0073 2,0023 2,0049 0,6950 0,7773 0,7618 0,8027 0,7612 0,6814 0,5078 0,4638 0,5135 34,476% 38,606% 38,050% 39,840% 38,020% 34,004% 25,298% 23,163% 25,612% Carrageenan (g) Average Carrageenan (g) SD Carrageenan (g) Yield (% dw) Average Yield (% dw) SD Yield (% dw) Hot Water Extraction (90 ºC) Algae mass (g) Algae Time Test 37,044% Chondrus crispus 60 min 30 min 10 min 0,7484 0,0616 37,288% 2,986% 2,241% 0,7447 0,0437 1,332% 0,4950 0,0272 24,691% 100 Table C.2.3. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HWE for the control extraction (60, 30 and 10 minutes) of alga G. pistillata Note: For the sample test 2 at 10 minutes, the polysaccharide clued to the recipient, and it was not possible to remove in a way that didn’t compromise the weight. Appendix D - Yield of the “less sulphated” K2-carrageenan from HAE (SE and Control) Calculation example of yield: Sample: Test 1 of 60 min – Mastocarpus stellatus YY = (mass precipitated · total mass after HAE) / (total mass of algae · mass after HAE used for the precipitation) (5) YY = (1,7953 · (2,4899 + 2,4895 + 2,3826))/((2,004 + 2,007 + 2,0015) · 5,0166)) · 100 = 43,82 % d.w. Using the formula for the propagation of error: SD YY=YY⋅((SD mass precipitated / mass precipitated)2+ (SD total mass after HAE / total mass after HAE)2 + (SD total mass of algae / total mass of algae )2 + (SD mass after HAE used for the precipitation / mass after HAE used for the precipitation )2 )1/2 And, knowing that the mass precipitated and the mass after HAE used for precipitation didn’t have a standard deviation error, the fraction equals zero, the equation looks like this: SD YY = 43,82 ⋅((0)2+ (0,06183 / (2,4899 + 2,4895 + 2,3826) )2 + (0,00275/ (2,004 + 2,007 + 2,0015))2 + (0)2 )1/2 = 0,369 % d.w. 1 2 3 1 2 3 1 2 3 2,0043 2,0012 2,0192 2,0265 2,0118 2,0046 2,0176 2,0293 2,0158 0,3265 0,4004 0,4081 0,3988 0,4329 0,3401 0,2619 - 0,2632 16,290% 20,008% 20,211% 19,679% 21,518% 16,966% 12,981% - 13,057% Carrageenan (g) Average Carrageenan (g) SD Carrageenan (g) Yield (% dw) Average Yield (% dw) SD Yield (% dw) Hot Water Extraction (90 ºC) Algae mass (g) Algae Time Test Gigartina pistillata 60 min 30 min 10 min 0,3906 0,0469 19,388% 2,290% 0,2626 0,0009 13,019% 0,054% 0,3783 0,0451 18,836% 2,208% 101 1.Sequential Extraction Table D.1.1. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HAE for the sequential extraction (60, 30 and 10 minutes) of alga M. stellatus Note: The chemical test didn’t show any carrageenans on the 10 min extract, so the yield doesn’t correspond to carrageenan. Table D.1.2. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HAE for the sequential extraction (60, 30 and 10 minutes) of alga C. crispus Table D.1.3. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HAE for the sequential extraction (60, 30 and 10 minutes) of alga G. pistillata 1 2 3 1 2 3 1 2 3 2,004 2,007 2,0015 2,0076 2,0083 2,001 2,0322 2,0168 2,0175 Mastocarpus stellatus 60 min 30 min 10 min Algae Time Test Initial dry algae mass (g) 2,4899 2,4895 2,3826 2,8862 2,6986 2,5324 1,7948 1,6606 1,7625 Mass after HAE (g) Hot Alkaline Extraction NaOH 3% (w/v) (90 ºC) Precipitation Total mass (g) Carrageenan precipitate (g) Yield (% dw) SD yield (% dw) SD mass after HAE (g) SD alga mass (g) 5,0551 0,1216 2,069% 36,557% 0,369% 0,798% 0,0279% 5,0202 1,3604 5,0166 1,7953 43,820% 0,06183 0,00275 0,17701 0,00403 0,07004 0,00870 1 2 3 1 2 3 1 2 3 2,0101 2,018 2,0267 2,0076 2,0083 2,001 2,0052 2,0044 2,0166 Chondrus crispus 60 min 30 min 10 min Algae Time Test Initial dry algae mass (g) 2,8926 2,6062 2,7683 3,0070 2,8799 2,4134 2,5872 2,0315 2,8086 Mass after HAE (g) Hot Alkaline Extraction NaOH 3% (w/v) (90 ºC) Precipitation Total mass (g) Carrageenan precipitate (g) Yield (% dw) SD yield (% dw) SD mass after HAE (g) SD alga mass (g) 0,166% 0,188% 0,2142 9,537% 4,987% 13,188% 0,1402 0,0723 2,0073 2,0001 2,0019 0,711% 0,14362 0,31255 0,40036 0,00830 0,00403 0,00682 1 2 3 1 2 3 1 2 3 2,0043 2,031 2,0618 2,0157 2,0045 2,0031 2,0019 2,0115 2,0123 60 min 30 min 10 min Gigartina pistillata Algae Time Test Initial dry algae mass (g) 1,8314 1,7286 1,1391 1,8422 1,9988 2,1694 1,6494 1,7386 2,0993 Mass after HAE (g) Hot Alkaline Extraction NaOH 3% (w/v) (90 ºC) Precipitation Total mass (g) Carrageenan precipitate (g) Yield (% dw) SD yield (% dw) SD mass after HAE (g) SD alga mass (g) 2,5928 1,3496 4,20% 1,61% 1,06% 52,703% 59,221% 24,499% 4,5518 4,3688 5,0166 3,1126 0,37358 0,16365 0,23821 0,02877 0,00691 0,00579 102 2.Control Table D.2.1. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HAE for the control extraction (60, 30 and 10 minutes) of alga M. stellatus Note: The chemical test didn’t show any carrageenans on the 10 min extract, so the yield doesn’t correspond to carrageenan. Table D.2.2. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HAE for the control extraction (60, 30 and 10 minutes) of alga C. crispus 1 2 3 1 2 3 1 2 3 Algae Time Test Mastocarpus stellatus 60 min 30 min 10 min 2,0111 2,0180 2,0008 2,0314 2,0234 2,0048 2,0069 2,0169 2,0055 2,0095 2,0086 2,0094 2,0167 2,0013 2,0280 2,0194 2,0194 2,0056 2,0120 2,0487 2,0204 1,9915 2,0097 2,0340 2,0016 2,0014 2,0011 1,7373 1,7409 1,6992 2,9664 2,4320 2,9625 1,9413 2,1652 1,0088 3,1357 2,9100 2,8512 2,7631 2,7377 2,6870 2,5981 3,0231 3,2390 1,6485 1,2648 1,7254 1,5231 1,6041 1,5887 1,9611 2,1966 2,1707 Hot Alkaline Extraction NaOH 3% (w/v) (90 ºC) Initial mass (g) (1) 0,6741 Algae mass (g) Mass (g) Precipitation Carrageenan precipitate (g) (1) 2,0019 0,0800 2 2,0034 0,6453 Yield (% dw) SD Yield (% dw) Hot Alkaline Extraction NaOH 3% (w/v) (90 ºC) 0,136% 0,648% 3,28% 17,598% 0,8115 Initial mass (g) (2) Carrageenan precipitate (g) (2) TOTAL Initial mass (g) TOTAL Carrageenan precipitate (g) 0,6741 4,3606 0,7315 6,3625 2 27,327% 2,0034 0,6453 28,940% 0,02311 0,30742 0,61332 0,00866 0,01365 0,00622 SD alga mass (g) SD mass after HAE (g) 1 2 3 1 2 3 1 2 3 Algae Time Test Chondrus crispus 60 min 30 min 10 min 2,0095 2,0086 2,0094 2,0167 2,0013 2,0280 2,0194 2,0194 2,0056 2,0120 2,0487 2,0204 1,9915 2,0097 2,0340 2,0016 2,0014 2,0011 3,1357 2,9100 2,8512 2,7631 2,7377 2,6870 2,5981 3,0231 3,2390 1,6485 1,2648 1,7254 1,5231 1,6041 1,5887 1,9611 2,1966 2,1707 Hot Alkaline Extraction NaOH 3% (w/v) (90 ºC) Initial mass (g) (1) Algae mass (g) Mass (g) Precipitation Carrageenan precipitate (g) (1) 0,5205 2 2,0077 0,8764 2,0023 0,4210 Yield (% dw) SD Yield (% dw) Hot Alkaline Extraction NaOH 3% (w/v) (90 ºC) 1,09% 0,334% 1,14% 2,7897 30,821% 7,0081 3,3102 63,967% 0,4210 Initial mass (g) (2) Carrageenan precipitate (g) (2) TOTAL Initial mass (g) TOTAL Carrageenan precipitate (g) 5,0081 0,8764 64,433% 2,0077 2,0023 SD alga mass (g) 0,15019 0,03874 SD mass after HAE (g) 0,32609 0,00049 0,01340 0,00797 103 Table D.2.3. Yield and Standard Deviation (SD) of the K2-carrageenan extract from the HAE for the control extraction (60, 30 and 10 minutes) of alga G. pistillata Note: The chemical test didn’t show any carrageenans on the 10 min extract, so the yield doesn’t correspond to carrageenan. Appendix E – FTIR spectra of the extracts from CWE, HWE and HAE E.1.Extracts from the CWE Figure E.1.1. FTIR spectra from the CWE K2-carrageenan extracts. 1 2 3 1 2 3 1 2 3 Algae Time Test Gigartina pistillata 60 min 30 min 10 min 2,0120 2,0487 2,0204 1,9915 2,0097 2,0340 2,0016 2,0014 2,0011 1,6485 1,2648 1,7254 1,5231 1,6041 1,5887 1,9611 2,1966 2,1707 Hot Alkaline Extraction NaOH 3% (w/v) (90 ºC) Initial mass (g) (1) Algae mass (g) Mass (g) Precipitation Carrageenan precipitate (g) (1) 2,0011 2 1,2345 0,5787 0,4189 2 Yield (% dw) SD Yield (% dw) Hot Alkaline Extraction NaOH 3% (w/v) (90 ºC) Initial mass (g) (2) Carrageenan precipitate (g) (2) TOTAL Initial mass (g) TOTAL Carrageenan precipitate (g) 2,51% 0,244% 0,451% 47,084% 24,994% 22,076% 2,0500 0,7171 2 4,0511 2 1,2345 1,2958 0,4189 SD alga mass (g) SD mass after HAE (g) 0,24674 0,04301 0,12914 0,01923 0,02132 0,00025 110 Figure H.1.1.3. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the shear rate (ɣ), at a 25 ºC and 1 Hz, of K2-carrageenan samples (1 hour and 30 min sequential water extraction from Mastocarpus stellatus) , in a 2 wt.% + 1 M NaCl solution. 1.2. Liquids Cold Water Extraction The extract didn´t have K2-carrageenans. Hot Water Extraction Figure H.1.2.4. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples (1 hour and 30 min control water extraction from Mastocarpus stellatus) , in a 2 wt.% + 1 M NaCl solution. 111 Alkaline Extraction Figure H.1.2.5. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples (1 hour and 30 min control alkaline extraction (top line); 1 hour and 30 min sequential alkaline extraction (bottom line) from Mastocarpus stellatus) , in a 2 wt.% + 1 M NaCl solution. 112 H.2 .Chondrus crispus 2.1.Gels Hot Water Extraction Figure H.2.1.1. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) during cooling from 85 ºC to 25 ºC of K2-carrageenan samples (1 hour and 30 min sequential and 30 min control water extraction from Chondrus crispus ), in a 2 wt.% + 1 M NaCl solution. 113 Figure H.2.1.2. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples (1 hour and 30 min sequential and 30 min control water extraction from Chondrus crispus ), in a 2 wt.% + 1 M NaCl solution. 114 Figure H.2.1.3. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the shear rate (ɣ), at a 25 ºC and 1 Hz, of K2-carrageenan (1 hour and 30 min sequential and 30 min control water extraction from Chondrus crispus ), in a 2 wt.% + 1 M NaCl solution. 2.2.Liquids Cold Water Extraction Figure H.2.2.1. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples (16h overnight cold water extraction from Chondrus crispus ), in a 2 wt.% + 1 M NaCl solution. 115 Hot Water Extraction Figure H.2.2.2. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples ( 10 min and 30 min control and 10 min sequential water extraction from Chondrus crispus) , in a 2 wt.% + 1 M NaCl solution. Alkaline Extraction There is no data, once the mass extracted wasn’t enough for these tests. 116 H.3. Gigartina pistillata 3.1.Gel Water Extraction Figure H.3.1.1. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) during cooling from 80 ºC to 25 ºC of K2-carrageenan samples (1 hour and 30 min sequential water extraction from Gigartina pistillata ), in a 2 wt.% + 1 M NaCl solution. Figure H.3.1.2. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples (1 hour and 30 min sequential and 30 min control water extraction from Gigartina pistillata ), in a 2 wt.% + 1 M NaCl solution. 117 Figure H.3.1.3. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the shear rate (ɣ), at a 25 ºC and 1 Hz, of K2-carrageenan (1 hour and 30 min sequential and 30 min control water extraction from Gigartina pistillata ), in a 2 wt% + 1 M NaCl solution. 3.2.Liquids Cold Water Extraction Figure H.3.2.1. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples (16h overnight cold water extraction from Gigartina pistillata) , in a 2 wt.% + 1 M NaCl solution. 118 Hot Water Extraction Figure H.3.2.2. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples (1 hour, 30 min and 10 min control and 10 min sequential water extraction from Gigartina pistillata) , in a 2 wt.% + 1 M NaCl solution. 119 Hot Alkaline Extraction Figure H.3.2.3. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples (1 hour and 30 min control extraction; 1 hour, 30 min and 10 min sequential alkaline extraction) from Gigartina pistillata) , in a 2 wt.% + 1 M NaCl solution. SE HAE 126 J.3. Alga Mastocarpus 3.1.Gel Figure J.3.1.1. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) during cooling from 85 ºC to 25 ºC of K2-carrageenan samples, from Mastocarpus , with 0h, 6h, 12h and 16h ultrasound, in a 1 wt.% + 0,1 M KCl solution. Figure J.3.1.2. Temperature dependence of the thickness (mm) of K2-carrageenan samples, from Mastocarpus , with 0h, 6h,12h and 16h of ultrasound, in a 1 wt.% + 0,1 M KCl solution, during rheological testing. 127 Figure J.3.1.3. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples, from Mastocarpus , with 0h, 6h, 12h and 16h ultrasound, in a 1 wt.% + 0,1 M KCl solution. Figure J.3.1.4. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the shear strain (%), at a 25 ºC and 1 Hz, of K2-carrageenan from Mastocarpus , with 0h, 6h, 12h and 16h of ultrasound, in a 1 wt.% + 0,1 M KCl solution. 128 3.2.Liquid Figure J.3.2.1. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 50,3 % strain, of K2-carrageenan samples from Mastorcapus with 20h of ultrasound, in a 1 wt% + 0,1 M KCl solution. 129 J.4. Alga BAT 18-09 4.1.Gel Figure J.4.1.1. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) during cooling from 85 ºC to 25 ºC of K2-carrageenan samples, from BAT 18-09, with 0h, 6h, 12h and 14h ultrasound, in a 1 wt.% + 0,1 M KCl solution. Figure J.4.1.2. Temperature dependence of the thickness (mm) of K2-carrageenan samples, from BAT, with 0h, 6h,12h and 14h of ultrasound, in a 1 wt.% + 0,1 M KCl solution, during rheological testing. 130 Figure J.4.1.3. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples, from BAT 18-09, with 0h, 6h, 12h and 14h ultrasound, in a 1 wt.% + 0,1 M KCl solution. Figure J.4.1.4. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the shear strain (%), at a 25 ºC and 1 Hz, of K2-carrageenan from BAT 18-09, with 0h, 6h, 12h and 14h of ultrasound, in a 1 wt.% + 0,1 M KCl solution. 131 4.2.Liquid Figure K.4.2.1. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 50,3 % strain, of K2-carrageenan samples from BAT 18-09 with 16h of ultrasound, in a 1 wt.% + 0,1 M KCl solution. J.5. Alga C-25 5.1.Gel Figure J.5.1.1. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) during cooling from 85 ºC to 25 ºC of K2-carrageenan samples, from C-25, with 0h and 6h ultrasound, in a 1 wt.% + 0,1 M KCl solution. 132 Figure J.5.1.2. Temperature dependence of the thickness (mm) of K2-carrageenan samples, from C-25, with 0h and 6h of ultrasound, in a 1 wt.% + 0,1 M KCl solution, during rheological testing. Figure J.5.1.3. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 0,1 % strain, of K2-carrageenan samples, from C-25, with 0h and 6h ultrasound, in a 1 wt.% + 0,1 M KCl solution. Figure J.5.1.4. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the shear strain (%), at a 25 ºC and 1 Hz, of K2-carrageenan from C-25, with 0h and 6h of ultrasound, in a 1 wt.% + 0,1 M KCl solution. 133 5.2.Liquid Figure J.5.2.1. Storage modulus (G′) (solid symbols) and loss modulus (G′′) (empty symbols) as a function of the oscillation frequencies (ω), at a 25 ºC with a 50,3 % strain, of K2-carrageenan samples from C-25 with 12h, 16h and 20h of ultrasound, in a 1 wt.% + 0,1 M KCl solution.