Impact of pre-treatment strategies for enhance conversion of Irish Brown Seaweed into high value ingredients using a biorefinery approach.

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Yiting Han, Xinyu Tan, Stephen Fitzpatrick, Henry Lyons, Xianglu Zhu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5696543/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This paper investigates the impact of different pre-treatments on the bio-refinery processes of wild-harvested Irish brown seaweeds, specifically Ascophyllum nodosum and Fucus vesiculosus . Employing a combination of mechanical and chemical methodologies, including drying, soaking, and the application of specific reagents, we aimed to optimize the extraction of valuable polysaccharides such as laminarin, fucoidan, and alginate, alongside protein recovery. The research highlighted the significant influence of pre-treatment methods on extraction efficiencies and polysaccharide purity, indicating that drying is beneficial for improving the purity and yield of laminarin and fucoidan, with laminarin purity as high as 61.46% in Ascophyllum nodosum . The study also demonstrates a complex interplay in alginate extraction across different treatments, with fresh treatments achieving up to 93.15% purity in Fucus vesiculosus. FT-IR provided insight into structural alterations and functional group exposure of extracted polysaccharides, indicating the potential of pre-treatment strategies in enhancing the yield and quality of bioactive compounds. These findings advance our understanding of seaweed bio-refinery processes and underscore the importance of pre-treatment selection in maximizing the sustainable utilization of marine resources for pharmaceutical, nutraceutical, and agricultural applications. Ascophyllum nodosum Fucus vesiculosus Biorefinery Laminarin Fucoidan Alginate Figures Figure 1 Figure 2 Figure 3 1. Introduction The exploration of renewable marine resources for sustainable and innovative applications has catapulted the science of biorefinery, particularly within the domain of seaweed research, into the forefront of contemporary scientific inquiry. Seaweeds, as renewable marine resources, have garnered extensive research interest for their bioactive compounds, particularly polysaccharides, in the fields of food, pharmaceuticals, and agriculture. These organisms, fundamental to marine ecosystems, offer a cornucopia of bioactive compounds, including polysaccharides, proteins, and minerals, underscoring their potential in a myriad of biotechnological applications ranging from nutraceuticals to bioenergy. Ascophyllum nodosum and Fucus vesiculosus , two widely studied brown seaweeds, have attracted attention for their unique bioactive compounds. Ascophyllum nodosum , commonly known as rockweed, is widespread on the north-eastern coast of North America and the northwest coast of Europe. This brown seaweed is a rich source of various bioactive compounds including phlorotannin and unique polysaccharides such as alginic acid, fucoidans, and laminarin. Studies have demonstrated the significant bioactivity of A. nodosum polysaccharides, including anti-inflammatory, antioxidant, and gut microbiota modulation effects (Shukla et al., 2019; Wang et al., 2023). Fucus vesiculosus , also known as bladderwrack, is rich in bioactive components, including polysaccharides and phenolic compounds. These components have shown potential in preventing or treating metabolic syndrome and related diseases ​(Keleszade et al., 2021). The polysaccharides from this seaweed have demonstrated potential to regulate blood sugar and reduce blood lipids, underscoring their potential applications in food and pharmaceutical industries ​(Keleszade et al., 2021). Recent advancements in biopolymer research have significantly expanded the applications of marine-derived polysaccharides such as laminarin, fucoidan, and alginate. As key components in seaweed-based biorefinery systems, these compounds can be efficiently extracted and valorized through integrated processing methods that aim to maximize resource utilization and minimize waste. Biorefinery strategies enable the cascading recovery of multiple bioactive fractions, such as polysaccharides and proteins, from seaweed biomass, enhancing the economic and environmental sustainability of seaweed-derived products. Consequently, these polysaccharides are increasingly utilized in various sectors including healthcare, food, and biotechnology due to their unique bioactive properties and potential to be derived from environmentally friendly, renewable sources. The implementation of biorefinery approaches not only supports the development of high-value products but also contributes to a circular bioeconomy by transforming seaweed into a versatile platform for sustainable innovation. Laminarin is explored for its potential in developing functional foods and nutraceuticals, while also being studied for its biodegradable and biocompatible nature in biomedical applications​ (Karuppusamy et al., 2022). Fucoidan has been recognized for its roles in regenerative medicine, drug delivery systems, and as a functional ingredient in the food industry due to its diverse biological actions​ (Anisha et al., 2022; Zayed and Ulber, 2020) ​. Furthermore, alginate beads are prominently used in drug delivery systems and tissue engineering, where their ability to form hydrogels is utilized to encapsulate drugs or cells, offering controlled release and protective environments​ (Mollah et al., 2021; Szekalska et al., 2016)​. These applications not only demonstrate the polysaccharides’ versatility but also their potential to contribute to sustainable practices and advanced medical therapies. This study aims to explore the biorefinery methods of polysaccharides from Ascophyllum nodosum and Fucus vesiculosus , their bioactivities, and their potential applications in agriculture and medicine to unlock their full potential. These range from promoting plant growth and health in agriculture to offering therapeutic benefits in medicine, such as antioxidant, anti-inflammatory, and antimicrobial effects. By conducting a more in-depth investigation of the polysaccharide components in these two brown seaweeds, we anticipate developing new functional foods, pharmaceuticals, and biostimulants to meet the growing health and agricultural needs. 2. Method & Material 2.1 Seaweed Ascophyllum nodosum and Fucus vesiculosus were freshly harvested from Co. Kerry, Ireland in March 2023. Fresh seaweed samples were transported to the lab, rinsed with tap water to remove surface contaminants and sea salt, and then dried to a length of 1 to 2 cm. A portion of the seaweed was stored at -20°C, one portion was oven-dried, and another portion was soaked in tricarballylic acid and salt solution. All samples were stored at -30°C before extraction. All reagents, including alginic acid, beta-glucan, fucose and laminarin, acetone, potassium bromide, were purchased from Sigma-Aldrich, USA. Maximum recovery diluent CM0733 (MRD) was purchased from Oxoid, UK. 2.2 Biorefinery process 2.2.1 Pre-treatment The seaweed biorefinery process designed in this study was illustrated in Fig. 1 modified by (Zhu et al., 2023a). It comprises two primary steps for cascading extracting laminarin/fucoidan and alginate, resulting in the recovery of enriched proteins in the final residue. One of the pre-treatment methods involves soaking the seaweed in a solution of 4.5% NaCl and 1.5% tricarballylic acid for two weeks, compared to the fresh and dried samples. 2.2.2 Laminarin/fucoidan extraction process Fresh and soaked Ascophyllum nodosum and Fucus vesiculosus (400 g) and dry Ascophyllum nodosum and Fucus vesiculosus (40 g) were mixed with 600 mL of a tricarballylic acid in a water bath at 70°C for 2.5 h, following a modified conventional extraction method described by (Zhu et al., 2023b). The fucoidan and laminarin mixture was obtained by adding 1.8 L of ethanol to the supernatant at a 1:3 (v/v) ratio, and the resulting solid was subjected to freeze drying. 2.2.3 Alginate extraction process The residual biomass, after the recovery of laminarin/fucoidan, was mixed with 800 mL of 2% (w/v) sodium carbonate in a water bath at 60°C for 3 h, using a solid-to-solvent ratio of 1:20 (w/v). Alginate dissolved in the supernatant, and the addition of recovered ethanol led to alginate precipitation, followed by freeze-drying. Concurrently, the pellet contained a significant quantity of proteins. 2.2.4 Ethanol recovery process The liquid obtained following the recovery of fucoidan and laminarin underwent ethanol recovery using a rotary evaporator (Rotavapor R-300, Buchi, Switzerland), and the reclaimed ethanol was used for alginate extraction. The ethanol-recovered water extracts were observed to contain a variety of polyphenols and pigments. 2.3 Laminarin, fucoidan and alginate measurements using high-performance liquid chromatography - refractive index detector (HPLC-RI) The methodology for quantifying laminarin fucoidan and alginate was refined based on the research conducted by Zhang and Row (2015). In this study, a high-performance liquid chromatography (HPLC) approach was utilized, utilizing an Agilent 1200 LC system (manufactured by Agilent Technologies, located in Santa Clara, California, USA). This HPLC apparatus was outfitted with a refractive index detector and was supported by a guard column (OHpak SB-G 6B, 8 x 50 mm) along with a Shodex OHpak SB-804 HQ carbohydrate column. This column featured 6% cross-linking and dimensions of 8 x 300 mm (length x inner diameter), produced by Shodex, Japan. Preparation of all samples was standardized at a concentration of 2 mg/mL in the chosen running solvent, followed by filtration through 0.45 µm Econo Filters (made of PTFE, supplied by Agilent) for clarity and purity. 2.4 Fourier-transform infrared spectroscopy (FT-IR) measurements FT-IR spectra of the samples were recorded to identify potential variations in functional groups compared to the standard. The samples were positioned on the surface of a diamond crystal attenuated total reflectance (ATR) accessory (iD7 ATR, Thermo Scientific, Madison, WI, USA), and spectral measurements were conducted with a Fourier transform mid-infrared spectrometer (Nicolet™ iS5, Thermo Scientific, Madison, WI, USA). Single-beam spectra were recorded in transmission mode over the range of 4000–400 cm − 1 with 64 scans at a resolution of 0.3 cm − 1 , and the differences were documented. Air blank background calibration was conducted prior to each measurement. Each measurement consisted of 64 scans to obtain averaged spectral data. Spectral data acquisition was supervised using the provided OMNIC software version 9.2.98 (Thermo Fisher Scientific Inc., USA)(Rajauria et al., 2023). 2.5 DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of Laminarin/fucoidan DPPH was evaluated to showcase the antioxidant activities. Briefly, Seaweed (macroalgae) extracts as samples and ascorbic acid as a positive control were prepared to 1 mg/mL in 0.1 M citrate phosphate buffer with 0.3% of Triton X-100. The samples were prepared in triplicates. Then, 10 µL of a 2 mM methanolic DPPH solution was added to each well. Further, the reaction mixture was incubated at room temperature in the dark for 30 min and read against the blank at 515 nm before and after the reaction with the DPPH solution using a UV-Vis spectrophotometer. The inhibition percentage of DPPH scavenging activity was calculated using Eq. ( 1 ) below. $$\:DPPH\:radical\:scavenging\:activity\:\left(\%\right)=\frac{{Abs}_{Blank}-{Abs}_{Sample}}{{Abs}_{Blank}}⨯100$$ 1 where “Abs Blank ” is the absorbance of the Blank (DPPH solution without sample/standard), “Abs Sample ” is the absorbance of the test sample (DPPH solution plus test sample/standard). 2.6 Sodium alginate characterisation 2.6.1 Preparation of sodium alginate/Ca + 2 hydrogel beads CaCl 2 was added to the sodium alginate (SA) solution at a rate of 12.8 g per 100 mL. SA solutions (2 g per 100 mL) were prepared, allowed to fully hydrate by storing them at 4°C for 24 hours, and degassed using ultrasound. Beads were created by slowly dispensing 20 mL of the SA solution into 160 mL of CaCl 2 solution using a 1.2 mm (18½ G) needle. The beads were left in a gelling bath to solidify at room temperature (20 ± 2°C) for 30 minutes, then collected by filtration through a plastic mesh and washed three times with deionized water. 2.6.2 Textural properties of the beads Textural properties of the beads was evaluated by Lozano-Vazquez et al (2015). The primary textural properties, including hardness, springiness, and cohesiveness of the hydrogel calcium alginate beads, were assessed using a stable micro systems texturometer model TA-XT2i (Texture Technologies Corp., White Plains, NY, USA) fitted with a 30 kg load cell. To ensure reliable test reproducibility, given the particulate nature of the beads, a cylindrical steel probe with a substantial contact area (36 mm in diameter) was employed. Measurements were conducted at room temperature (20 ± 2°C) using 30 g of hydrogel calcium alginate beads positioned on a stationary glass plate beneath the probe. Automatic detection of probe-bead contact was performed with a contact force of 0.005 N. The samples underwent 30% compression through two cycles, each at a consistent crosshead velocity of 1.0 mm/s. Textural property values were acquired using the Texture Expert Software for Windows, Version 3.2, integrated with the equipment. 2.7 Protein content in residues The protein content (%) of the freeze-dried seaweed residues was determined by weighing and then analyzed using a nitrogen analyzer (FP-328, Leco Instrument, Leco Corporation, USA) following the Dumas principle (method 968.06, Official Methods of Analysis of AOAC International, 16th edition, Arlington, VA, USA: AOAC International, 1995) with a conversion factor of 6.25. 2.8 Total polyphenol content (TPC) A 100 µL portion of the extracts was mixed with 2mL 2% (w/v) Na 2 CO 3 and allowed to mix for 2 minutes at room temperature. Subsequently, 100 µL of Folin–Ciocalteu's reagent 1 M was added. The samples were kept in the dark at room temperature for 30 minutes, and the absorbance was measured at 720 nm. A sample blank (containing only solvent without a sample) was included. Gallic acid served as the standard, and the measured absorbance was converted to gallic acid equivalents using a calibration curve constructed with gallic acid. The calibration curve was generated by dissolving gallic acid in the same solvent as the extracts (either methanol or ethyl acetate) within a concentration range of 0 to 0.5 mg mL⁻¹. 2.9 Statistical analysis Each pre-treatment condition was replicated three times. Subsequently, each sample was analyzed three times to ensure the accuracy and reliability of the results. The statistical analysis of experimental data was conducted using one way analysis of variance (ANOVA), facilitated by the Statistical Package for the Social Sciences (SPSS) software, version 20.0.0 (IBM, U.S.A.). This approach allowed for the identification of significant differences between the treatment groups, with a predefined significance threshold set at P < 0.05. Data management, including the organization and preliminary analysis, as well as graphical representations of the findings, were handled using Microsoft Excel. For more complex graphical representations, MATLAB (version, company, America) software was employed. 3. Results & Discussion 3.1 Biorefinery efficiency Table 1 provides a comparative overview of the purity rates of laminarin, fucoidan, and alginate under different pre-treatment conditions. For laminarin, fresh treatment yielded a laminarin purity of 48.32% in Ascophyllum nodosum , contrasting with a much lower purity of 10.98% in Fucus vesiculosus , indicating inherent differences in their biological composition or structure that affect compound extraction. The dry treatment significantly boosted laminarin purity in Ascophyllum nodosum to 61.46%, while also elevating Fucus vesiculosus ' purity to 39.63%. This suggests that drying facilitates the breakdown of cellular barriers, making laminarin more accessible (Kadam et al., 2015). Interestingly, the soaked treatment drastically reduced laminarin purity in A. nodosum to a mere 0.55%, while F. vesiculosus , experienced an increase to 38.96%, highlighting a species-specific response that may involve differences in water absorption or laminarin solubilization. Table 1 Purity (%) of laminarin, Fucoidan and sodium alginate Sample Purity Rates(%) Laminarin Fucoidan Alginate Fresh Asco 48.32 13.45 66.33 Fresh Fucus 10.98 13.81 93.15 Dry Asco 61.46 43.63 68.31 Dry Fucus 3.55 39.63 65.28 Soaked Asco 0.55 12.83 67.01 Soaked Fucus 27.01 14.16 75.40 Table 2 DPPH of laminarin and sodium alginate Sample DPPH (%) Fresh Asco 49.88 ± 3.79 Fresh Fucus 47.07 ± 1.16 Dry Asco 62.15 ± 0.56 Dry Fucus 26.69 ± 5.20 Socked Asco 30.14 ± 2.61 Socked Fucus 24.20 ± 2.97 The extraction of fucoidan presented a clear advantage with the drying process, where both A. nodosum and F. vesiculosus saw significant increases in purity, reaching 43.63% and 39.63%, respectively. This enhancement likely occurs from the dehydration effect altering the seaweed’s cell structure in a manner conducive to fucoidan release. Conversely, soaking had a detrimental impact, particularly on Fucus, where purity plummeted to 6.28%, indicating that fucoidan extractability is adversely affected by aqueous conditions, possibly due to degradation or leaching effects. Alginate extraction offered additional insights into the complexity of pre-treatment effects. Fresh samples showed high alginate purity, especially in Fucus at 93.15%, indicating a more readily extractable state in its natural form, possibly due to higher alginate content or structural differences. The drying process slightly increased alginate purity in Ascophyllum nodosum but decreased it in Fucus vesiculosus , suggesting a delicate balance between moisture content and alginate's extractability or stability. Soaking proved to be less beneficial for alginate extraction, particularly affecting F. vesiculosus , where purity decreased significantly to 42.11%. This could be attributed to the leaching of alginate into the soaking medium or alterations in its molecular configuration in the presence of excess water. 3.2 FT-IR analysis The raw FT-IR absorbance spectra of extracted sodium alginate and mixtures of laminarin and fucoidan collected over the wavelength range of 4000-400cm -1 are shown in Fig. 2 respectively, compared with the standard, Laminarin-S, Fucose-S, Glucan-S and Alginate-acid-s. The spectra exhibit an increase in absorbance intensity in the O-H stretching vibrations within the 3200–3600 cm⁻¹ range and C-H stretching vibrations around 2920 cm⁻¹ (Garza-Cervantes et al., 2019). This enhancement was particularly pronounced in samples subjected to soaking treatments, such as Soaked A. nodosum and Soaked F. vesiculosus , indicating a potential increase in hydroxyl and alkyl chain contents across both laminarin, fucoidan, and alginate samples. The elevated absorbance suggests that soaking methods may modify the water-binding capacity and hydrophobic interactions within these polysaccharides, enhancing their extraction yields. The analysis revealed a marked increase in absorbance at 1620 cm⁻¹ and 1420 cm⁻¹, indicative of carboxylate functional groups, across all processed samples (Frota et al., 2024). The increase was most significant in alginate samples undergoing soaking treatment, highlighting that the soaking process facilitates the exposure or increases the content of carboxylate groups, crucial for the functionality and solubility of alginate. The spectral analysis within the 1320 − 1260 cm⁻¹ and 1080 − 1030 cm⁻¹ regions, corresponding to C-O-C and C-O stretching vibrations, showed that processed samples displayed higher absorbance intensity than the standard references for laminarin, fucoidan, and alginate (Khalafu et al., 2017). This increase, especially notable in soaked samples, implies that soaking treatments may preserve or enhance the polysaccharide structures. Specifically, for fucoidan, the absorbance intensities at wavelengths indicative of sulfate ester groups (approximately 1250 cm⁻¹ and 820 cm⁻¹) were higher in samples processed through soaking treatments (Mohd Fauziee et al., 2021). This observation suggests an increase in sulfation levels or better preservation of sulfate ester groups, highlighting the specificity of soaking effects on fucoidan's structural components. The integrated spectroscopic analysis underscores the nuanced impact of soaking treatments on the extraction efficiency and structural preservation of laminarin, fucoidan, and alginate. The enhanced absorbance intensity in key functional groups across all polysaccharides suggests that soaking treatments can significantly improve yield and quality by altering the hydrophilic and hydrophobic balance and exposing more functional groups essential for their biological and physicochemical properties. Soaking treatments, particularly those applied to Socked A. nodosum and Socked F. vesiculosus , samples, have demonstrated superior performance in terms of extraction efficiency, outperforming fresh and dried samples. This indicates that soaking not only enhances the solubility of these polysaccharides but may also facilitate the optimization of the extraction process and enhance product quality through better exposure and preservation of essential functional groups. 3.3 Antioxidant activities of fucoidan and laminarin mixtures (DPPH) The antioxidant capacities of fucoidan and laminarin mixtures were evaluated using the DPPH assay, which revealed significant effects of the pre-treatment on antioxidant activity. For the mixtures extracted from A. nodosum , fresh samples showed an antioxidant activity of 49.88 ± 3.79%. This significantly increased to 62.15 ± 0.556% after drying (P < 0.05), suggesting that drying may help concentrate or alter the extraction efficiency of antioxidant compounds. However, the activity markedly decreased in soaked samples to 30.14 ± 2.61%, likely due to the leaching of antioxidant components or degradation of active compounds in aqueous conditions. According to Siriwardhana et al.(2003) and Liu and Ng (2000), a strong correlation (r = 0.971) was found between the DPPH radical-scavenging activities and total polyphenolic content, which may also explain dry samples have high antioxidant potential compounds that less antioxidant compounds leak with phenolic compounds. However, there are higher total polyphenolics content in the soaked samples’ water extracts with more antioxidant potential compounds leaking. Conversely, the mixtures extracted from F . vesiculosu displayed an antioxidant activity of 47.07 ± 1.162% in fresh samples, which significantly decreased after drying to 26.69 ± 5.1999%, and slightly further decreased in soaked conditions to 24.20 ± 2.968% (P < 0.01), which is consistent with the findings of Jiménez-Escrig et al (2001). This significant reduction could be associated with structural or compositional differences between the two types of seaweed, which may influence the stability and extractability of antioxidant compounds under different treatment conditions. These results indicate that the method of pre-treatment significantly impacts the antioxidant potential of fucoidan and laminarin mixtures. These variations highlight the necessity of optimizing processing conditions tailored to each seaweed type to maximize the recovery of beneficial antioxidants, crucial for potential applications in nutraceuticals and functional foods. 3.4 Properties of alginate (calcium alginate-modified beads) Table 3 presents the geometrical and textural characteristics of calcium alginate-modified beads with 3 different treatments of Ascophyllum nodosum and Fucus vesiculosus . Comparing the alginate properties of A. nodosum obtained through different treatments, it can be seen that three treatments exert a significant impact on three critical textural parameters: hardness, viscosity and chewiness (P 0.05). Table 3 The geometrical and textural characteristics of calcium alginate-modified beads Hardness Adhesiveness Springiness Cohesiveness Gumminess Chewiness Resilience Fresh Asco 170.726 ± 65.01 a -1.821 ± 0.611 b 0.74 ± 0.05 a 0.736 ± 0.042 a 126.813 ± 51.808 a 95.047 ± 43.056 a 0.411 ± 0.052 a Fresh Fucus 276.081 ± 25.132 b -2.374 ± 0.691 b 0.73 ± 0.042 a 0.714 ± 0.014 a 197.31 ± 19.309 b 143.986 ± 15.08 b 0.437 ± 0.021 a Dry Asco 192.318 ± 15.928 a -2.961 ± 0.881 b 0.788 ± 0.017 a 0.745 ± 0.005 a 143.198 ± 10.937 a 112.907 ± 10.467 ab 0.435 ± 0.006 a Dry Fucus 266.555 ± 9.152 b -5.816 ± 3.128 b 0.779 ± 0.044 a 0.74 ± 0.065 a 197.642 ± 23.386 b 154.555 ± 26.232 b 0.464 ± 0.068 a Socked Asco 191.501 ± 22.274 a -4.602 ± 1.617 b 0.795 ± 0.034 a 0.767 ± 0.017 a 146.752 ± 16.072 ab 117.046 ± 17.434 ab 0.395 ± 0.01 a Socked Fucus 228.596 ± 8.182 ab -33.184 ± 10.933 a 0.771 ± 0.009 a 0.749 ± 0.042 a 171.245 ± 12.969 ab 132.088 ± 10.482 ab 0.42 ± 0.043 a Note: Data in the same column with the same letter are not significantly different (P > 0.05); all data are the means from 3 replicates. The pronounced disparities in hardness and viscosity imply that the treatments can modulate the density and strength of cross-linkages amongst alginate molecules, subsequently altering the structural integrity and mechanical attributes of the modified beads. This observation is congruent with prior studies, which have posited that pretreatment conditions, including drying and soaking, can influence the physical and chemical properties of alginates—namely, their molecular weight distribution and solubility—thereby affecting their functional properties, such as gel formation capacity and adhesiveness (McHugh, 1987). The marked variation in chewiness further emphasizes the significant role of treatments in modulating the mechanical performance of the modified beads, an aspect crucial for their potential functional applications within the realms of food processing and biomedical endeavours. The lack of significant differences in adhesiveness, elasticity, cohesiveness, and resilience may reflect the insensitivity of these textural characteristics to variations in pretreatment conditions, or possibly, limitations inherent to the experimental design and statistical analysis employed. For example, an increase in the sample size or the adoption of more sensitive measurement techniques might unveil subtle variations in these attributes. 3.5 Protein/Fibre Figure 3 shows the protein content across different samples and treatments. An ANOVA test was conducted to compare the protein content (%) across different treatment methods for each freeze-dried seaweed residue and raw materials. The result indicated a significant difference (P < 0.05) in protein content across different treatments, which are critical for optimizing seaweed bio-refinery processes. The residue with dry treatment exhibited the highest protein contents among the tested conditions, particularly notable in Ascophyllum nodosum (Dry Asco) with an average protein content of 16.66%, suggesting a concentration effect due to moisture removal, beneficial for applications requiring high protein yields. Conversely, soaked treatments resulted in reduced protein levels, likely due to the acid used which causes protein molecules to leach (Kadam et al., 2017). 3.6 TPC Water extracts after extracting the mixture of laminarin/fucoidan were collected. The comparative study demonstrated significant differences in TPC across the pre-treatments for each type of seaweed, with soaked samples consistently exhibiting the highest phenolic content across both seaweed types. Specifically, Water extracts of Fucus vesiculosus treated through soaking showed a remarkable increase in TPC concentration, indicating an enhanced extraction of phenolic compounds due to the pre-treatment process. The increase could be a result of acidification disintegrating cell walls of phenolic compounds, which helps facilitate phenolic compounds the solubilization and diffusion during the extraction (Charoensiddhi et al., 2015). This is in stark contrast to the dry treatment, which generally resulted in the lowest TPC values among the three methods evaluated. This result is consistent with the previous reports by Gupta et al., (2011). For Ascophyllum nodosum , while the difference in TPC between treatments was less pronounced than in Fucus vesiculosus , the soaked treatment still emerged as the most effective method for maximizing phenolic content extraction. The fresh treatment showed intermediate results, and similar to Fucus vesiculosus , the dry treatment yielded the lowest TPC values. The water extracts of Fucus vesiculosus showed a considerable variation in TPC among treatments. In contrast, Ascophyllum nodosum 's TPC was not significantly affected by the treatments. 4. Conclusion The comprehensive analysis of different pre-treatment methods in this study revealed the complicate impacts these methods have on the extraction efficiencies and qualities of laminarin, fucoidan, and alginate from Ascophyllum nodosum and Fucus vesiculosus. The research demonstrates that drying pre-treatments significantly enhances the extraction of laminarin and fucoidan by facilitating the breakdown of cellular barriers, thereby making these bioactive compounds more accessible. Conversely, soaking pre-treatments exhibit species-specific responses, which affect the purity levels of both laminarin and alginate differently, indicating a complex interplay between seaweed species, water absorption, and polysaccharide solubilization dynamics. Additionally, the FT-IR analysis provides a molecular perspective, demonstrating how various pre-treatments can modify the structural integrity and functional group exposure of the target polysaccharides, influencing their potential bioactive applications. This comprehensive examination of pre-treatment effects not only advances our understanding of seaweed bio-refinery processes but also highlights the importance of selecting appropriate pre-treatment strategies to optimize the yield and bioactivity of extracted compounds, laying a foundation for future innovations in the sustainable utilization of marine resources for nutraceutical, pharmaceutical, and agricultural applications. Declarations CRedit authorship contribution statement Yiting Han: Conceptualization, Methodology, Formal analysis, Data curation, Writing - original draft. Xinyu Tan: Methodology, Data curation, Writing - original draft. Stephen Fitzpatrick: Methodology, Data curation, Writing - review & editing. Henry Lyons: Methodology, Data curation, Writing - review & editing. Xianglu Zhu: Conceptualization, Project administration, Formal analysis, Data curation, Writing - review & editing, Supervision. Brijesh K Tiwari: Conceptualization, Project administration, Funding acquisition, Formal analysis, Data curation, Writing - review & editing, Supervision. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Anisha, G.S., Padmakumari, S., Patel, A.K., Pandey, A., Singhania, R.R., 2022. Fucoidan from Marine Macroalgae: Biological Actions and Applications in Regenerative Medicine, Drug Delivery Systems and Food Industry. Bioengineering 9, 472. https://doi.org/10.3390/bioengineering9090472 Antioxidant Activity of Hizikia fusiformis on Reactive Oxygen Species Scavenging and Lipid Peroxidation Inhibition - Nalin Siriwardhana, K.-W. 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Food Research International, Microalgae and Seaweeds as Potential Source of Valuable Nutrients, Food Additives and Nutraceuticals for Human and Animal Consumption 99, 1021–1027. https://doi.org/10.1016/j.foodres.2016.07.018 Kadam, S.U., O’Donnell, C.P., Rai, D.K., Hossain, M.B., Burgess, C.M., Walsh, D., Tiwari, B.K., 2015. Laminarin from Irish Brown Seaweeds Ascophyllum nodosum and Laminaria hyperborea: Ultrasound Assisted Extraction, Characterization and Bioactivity. Marine Drugs 13, 4270–4280. https://doi.org/10.3390/md13074270 Karuppusamy, S., Rajauria, G., Fitzpatrick, S., Lyons, H., McMahon, H., Curtin, J., Tiwari, B.K., O’Donnell, C., 2022. Biological Properties and Health-Promoting Functions of Laminarin: A Comprehensive Review of Preclinical and Clinical Studies. Marine Drugs 20, 772. https://doi.org/10.3390/md20120772 Keleszade, E., Patterson, M., Trangmar, S., Guinan, K.J., Costabile, A., 2021. Clinical Efficacy of Brown Seaweeds Ascophyllum nodosum and Fucus vesiculosus in the Prevention or Delay Progression of the Metabolic Syndrome: A Review of Clinical Trials. Molecules 26, 714. https://doi.org/10.3390/molecules26030714 Khalafu, S.H.S., Wan Aida, W.M., Lim, S.J., Maskat, M.Y., 2017. Effects of deodorisation methods on volatile compounds, chemical properties and antioxidant activities of fucoidan isolated from brown seaweed (Sargassum sp.). Algal Research 25, 507–515. https://doi.org/10.1016/j.algal.2017.06.018 Liu, F., Ng, T.B., 2000. Antioxidative and free radical scavenging activities of selected medicinal herbs. Life Sciences 66, 725–735. https://doi.org/10.1016/S0024-3205(99)00643-8 Lozano-Vazquez, G., Lobato-Calleros, C., Escalona-Buendia, H., Chavez, G., Alvarez-Ramirez, J., Vernon-Carter, E.J., 2015. Effect of the weight ratio of alginate-modified tapioca starch on the physicochemical properties and release kinetics of chlorogenic acid containing beads. Food Hydrocolloids 48, 301–311. https://doi.org/10.1016/j.foodhyd.2015.02.032 McHugh, D.J.D. of C., 1987. Production, properties and uses of alginates. FAO Fisheries Technical Paper (FAO). Mohd Fauziee, N.A., Chang, L.S., Wan Mustapha, W.A., Md Nor, A.R., Lim, S.J., 2021. Functional polysaccharides of fucoidan, laminaran and alginate from Malaysian brown seaweeds (Sargassum polycystum, Turbinaria ornata and Padina boryana). International Journal of Biological Macromolecules 167, 1135–1145. https://doi.org/10.1016/j.ijbiomac.2020.11.067 Mollah, M.Z.I., Zahid, H.M., Mahal, Z., Faruque, M.R.I., Khandaker, M.U., 2021. The Usages and Potential Uses of Alginate for Healthcare Applications. Front. Mol. Biosci. 8. https://doi.org/10.3389/fmolb.2021.719972 Rajauria, G., Ravindran, R., Garcia-Vaquero, M., Rai, D.K., Sweeney, T., O’Doherty, J., 2023. Purification and Molecular Characterization of Fucoidan Isolated from Ascophyllum nodosum Brown Seaweed Grown in Ireland. Marine Drugs 21, 315. https://doi.org/10.3390/md21050315 Shukla, P.S., Mantin, E.G., Adil, M., Bajpai, S., Critchley, A.T., Prithiviraj, B., 2019. Ascophyllum nodosum-Based Biostimulants: Sustainable Applications in Agriculture for the Stimulation of Plant Growth, Stress Tolerance, and Disease Management. Front. Plant Sci. 10. https://doi.org/10.3389/fpls.2019.00655 Siriwardhana, N., Lee, K.-W., Jeon, Y.-J., Kim, S.-H., Haw, J.-W., 2003. Antioxidant Activity of Hizikia fusiformis on Reactive Oxygen Species Scavenging and Lipid Peroxidation Inhibition. Food sci. technol. int. 9, 339–346. https://doi.org/10.1177/1082013203039014 Szekalska, M., Puciłowska, A., Szymańska, E., Ciosek, P., Winnicka, K., 2016. Alginate: Current Use and Future Perspectives in Pharmaceutical and Biomedical Applications. International Journal of Polymer Science 2016, e7697031. https://doi.org/10.1155/2016/7697031 Wang, Lilong, Yan, C., Wang, Linlin, Ai, C., Wang, S., Shen, C., Tong, Y., Song, S., 2023. Ascophyllum nodosum polysaccharide regulates gut microbiota metabolites to protect against colonic inflammation in mice. Food Funct. 14, 810–821. https://doi.org/10.1039/D2FO02964B Zayed, A., Ulber, R., 2020. Fucoidans: Downstream Processes and Recent Applications. Marine Drugs 18, 170. https://doi.org/10.3390/md18030170 Zhang, H., Row, K.H., 2015. Extraction and Separation of Polysaccharides from Laminaria japonica by Size-Exclusion Chromatography. Journal of chromatographic science 53, 498–502. https://doi.org/10.1093/chromsci/bmu073 Zhu, X., Healy, L., Das, R.S., Bhavya, M.L., Karuppusamy, S., Sun, D.-W., O’Donnell, C., Tiwari, B.K., 2023a. Novel biorefinery process for extraction of laminarin, alginate and protein from brown seaweed using hydrodynamic cavitation. Algal Research 74, 103243. https://doi.org/10.1016/j.algal.2023.103243 Zhu, X., Healy, L., Wanigasekara, J., Zhao, M., Padamati, R.B., Karuppusamy, S., Curtin, J.F., Sivagnanam, S.P., Rai, D.K., Sun, D.-W., Tiwari, B.K., 2023b. Characterisation of laminarin extracted from brown seaweed Laminaria digitata , using optimized ultrasound- and ultrafiltration-assisted extraction method. Algal Research 75, 103277. https://doi.org/10.1016/j.algal.2023.103277 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5696543","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":402527064,"identity":"74acb2c5-010b-4c21-b733-74480b100078","order_by":0,"name":"Yiting Han","email":"","orcid":"","institution":"University College Dublin - National University of Ireland: University College Dublin","correspondingAuthor":false,"prefix":"","firstName":"Yiting","middleName":"","lastName":"Han","suffix":""},{"id":402527065,"identity":"42e2fc5f-9d7d-44d1-8359-b71d0e33676d","order_by":1,"name":"Xinyu Tan","email":"","orcid":"","institution":"Shanghai Institute of Quality Inspection and Technical Research","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Tan","suffix":""},{"id":402527066,"identity":"1bb6d0d3-b764-4da5-b732-8813c037d2bf","order_by":2,"name":"Stephen Fitzpatrick","email":"","orcid":"","institution":"Nutramara Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"","lastName":"Fitzpatrick","suffix":""},{"id":402527067,"identity":"204c62e5-eb5a-4fa5-a6c5-32b6167a1d85","order_by":3,"name":"Henry Lyons","email":"","orcid":"","institution":"Nutramara Co. Ltd","correspondingAuthor":false,"prefix":"","firstName":"Henry","middleName":"","lastName":"Lyons","suffix":""},{"id":402527068,"identity":"b12be168-19f0-4843-a56f-5371289780a8","order_by":4,"name":"Xianglu Zhu","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-6941-1471","institution":"East China University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Xianglu","middleName":"","lastName":"Zhu","suffix":""},{"id":402527069,"identity":"5fc00e25-2f39-4968-9a76-50e7f029b826","order_by":5,"name":"Brijesh K Tiwari","email":"","orcid":"","institution":"Teagasc Food Research Centre Ashtown","correspondingAuthor":false,"prefix":"","firstName":"Brijesh","middleName":"K","lastName":"Tiwari","suffix":""}],"badges":[],"createdAt":"2024-12-23 05:04:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5696543/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5696543/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74057277,"identity":"ca6f758a-9666-4e0f-98b0-b65d6ec3fdd3","added_by":"auto","created_at":"2025-01-17 10:41:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38983,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic flow chart of brown seaweed biorefinery process\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5696543/v1/d46ece0182e6e0af3ba6e9ad.png"},{"id":74057280,"identity":"2554c0d7-3bc2-4456-bb4d-d982b91f2fdc","added_by":"auto","created_at":"2025-01-17 10:41:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":147179,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR spectra (400–4000 cm−1) of the laminarin and alginate \u003ca href=\"https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/extract\" title=\"Learn more about extracts from ScienceDirect's AI-generated Topic Pages\"\u003eextracts\u003c/a\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5696543/v1/f12077fd5ac7a6cabbbf02be.png"},{"id":74057279,"identity":"1d02459e-a968-4bfc-a25b-9aece0520347","added_by":"auto","created_at":"2025-01-17 10:41:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":29545,"visible":true,"origin":"","legend":"\u003cp\u003eProtein contents (%, w/w) of residues after biorefinery process\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5696543/v1/fab4b4ac560600a37dd386ee.png"},{"id":76930006,"identity":"4f00be08-8f6d-4b4b-8b46-c5b8d1cb4052","added_by":"auto","created_at":"2025-02-22 14:29:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1129524,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5696543/v1/ba043de0-b5e9-45c5-bf11-0c6dd39d82f0.pdf"}],"financialInterests":"","formattedTitle":"Impact of pre-treatment strategies for enhance conversion of Irish Brown Seaweed into high value ingredients using a biorefinery approach.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe exploration of renewable marine resources for sustainable and innovative applications has catapulted the science of biorefinery, particularly within the domain of seaweed research, into the forefront of contemporary scientific inquiry. Seaweeds, as renewable marine resources, have garnered extensive research interest for their bioactive compounds, particularly polysaccharides, in the fields of food, pharmaceuticals, and agriculture. These organisms, fundamental to marine ecosystems, offer a cornucopia of bioactive compounds, including polysaccharides, proteins, and minerals, underscoring their potential in a myriad of biotechnological applications ranging from nutraceuticals to bioenergy. \u003cem\u003eAscophyllum nodosum\u003c/em\u003e and \u003cem\u003eFucus vesiculosus\u003c/em\u003e, two widely studied brown seaweeds, have attracted attention for their unique bioactive compounds.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAscophyllum nodosum\u003c/em\u003e, commonly known as rockweed, is widespread on the north-eastern coast of North America and the northwest coast of Europe. This brown seaweed is a rich source of various bioactive compounds including phlorotannin and unique polysaccharides such as alginic acid, fucoidans, and laminarin. Studies have demonstrated the significant bioactivity of \u003cem\u003eA. nodosum\u003c/em\u003e polysaccharides, including anti-inflammatory, antioxidant, and gut microbiota modulation effects (Shukla et al., 2019; Wang et al., 2023). \u003cem\u003eFucus vesiculosus\u003c/em\u003e, also known as bladderwrack, is rich in bioactive components, including polysaccharides and phenolic compounds. These components have shown potential in preventing or treating metabolic syndrome and related diseases ​(Keleszade et al., 2021). The polysaccharides from this seaweed have demonstrated potential to regulate blood sugar and reduce blood lipids, underscoring their potential applications in food and pharmaceutical industries ​(Keleszade et al., 2021).\u003c/p\u003e \u003cp\u003eRecent advancements in biopolymer research have significantly expanded the applications of marine-derived polysaccharides such as laminarin, fucoidan, and alginate. As key components in seaweed-based biorefinery systems, these compounds can be efficiently extracted and valorized through integrated processing methods that aim to maximize resource utilization and minimize waste. Biorefinery strategies enable the cascading recovery of multiple bioactive fractions, such as polysaccharides and proteins, from seaweed biomass, enhancing the economic and environmental sustainability of seaweed-derived products. Consequently, these polysaccharides are increasingly utilized in various sectors including healthcare, food, and biotechnology due to their unique bioactive properties and potential to be derived from environmentally friendly, renewable sources. The implementation of biorefinery approaches not only supports the development of high-value products but also contributes to a circular bioeconomy by transforming seaweed into a versatile platform for sustainable innovation. Laminarin is explored for its potential in developing functional foods and nutraceuticals, while also being studied for its biodegradable and biocompatible nature in biomedical applications​ (Karuppusamy et al., 2022). Fucoidan has been recognized for its roles in regenerative medicine, drug delivery systems, and as a functional ingredient in the food industry due to its diverse biological actions​ (Anisha et al., 2022; Zayed and Ulber, 2020) ​. Furthermore, alginate beads are prominently used in drug delivery systems and tissue engineering, where their ability to form hydrogels is utilized to encapsulate drugs or cells, offering controlled release and protective environments​ (Mollah et al., 2021; Szekalska et al., 2016)​. These applications not only demonstrate the polysaccharides\u0026rsquo; versatility but also their potential to contribute to sustainable practices and advanced medical therapies.\u003c/p\u003e \u003cp\u003eThis study aims to explore the biorefinery methods of polysaccharides from \u003cem\u003eAscophyllum nodosum\u003c/em\u003e and \u003cem\u003eFucus vesiculosus\u003c/em\u003e, their bioactivities, and their potential applications in agriculture and medicine to unlock their full potential. These range from promoting plant growth and health in agriculture to offering therapeutic benefits in medicine, such as antioxidant, anti-inflammatory, and antimicrobial effects. By conducting a more in-depth investigation of the polysaccharide components in these two brown seaweeds, we anticipate developing new functional foods, pharmaceuticals, and biostimulants to meet the growing health and agricultural needs.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Method \u0026 Material","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Seaweed\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cem\u003eAscophyllum nodosum\u003c/em\u003e and \u003cem\u003eFucus vesiculosus\u003c/em\u003e were freshly harvested from Co. Kerry, Ireland in March 2023. Fresh seaweed samples were transported to the lab, rinsed with tap water to remove surface contaminants and sea salt, and then dried to a length of 1 to 2 cm. A portion of the seaweed was stored at -20\u0026deg;C, one portion was oven-dried, and another portion was soaked in tricarballylic acid and salt solution. All samples were stored at -30\u0026deg;C before extraction. All reagents, including alginic acid, beta-glucan, fucose and laminarin, acetone, potassium bromide, were purchased from Sigma-Aldrich, USA. Maximum recovery diluent CM0733 (MRD) was purchased from Oxoid, UK.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Biorefinery process\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Pre-treatment\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe seaweed biorefinery process designed in this study was illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e modified by (Zhu et al., 2023a). It comprises two primary steps for cascading extracting laminarin/fucoidan and alginate, resulting in the recovery of enriched proteins in the final residue. One of the pre-treatment methods involves soaking the seaweed in a solution of 4.5% NaCl and 1.5% tricarballylic acid for two weeks, compared to the fresh and dried samples.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Laminarin/fucoidan extraction process\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFresh and soaked \u003cem\u003eAscophyllum nodosum\u003c/em\u003e and \u003cem\u003eFucus vesiculosus\u003c/em\u003e (400 g) and dry \u003cem\u003eAscophyllum nodosum\u003c/em\u003e and \u003cem\u003eFucus vesiculosus\u003c/em\u003e (40 g) were mixed with 600 mL of a tricarballylic acid in a water bath at 70\u0026deg;C for 2.5 h, following a modified conventional extraction method described by (Zhu et al., 2023b). The fucoidan and laminarin mixture was obtained by adding 1.8 L of ethanol to the supernatant at a 1:3 (v/v) ratio, and the resulting solid was subjected to freeze drying.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Alginate extraction process\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe residual biomass, after the recovery of laminarin/fucoidan, was mixed with 800 mL of 2% (w/v) sodium carbonate in a water bath at 60\u0026deg;C for 3 h, using a solid-to-solvent ratio of 1:20 (w/v). Alginate dissolved in the supernatant, and the addition of recovered ethanol led to alginate precipitation, followed by freeze-drying. Concurrently, the pellet contained a significant quantity of proteins.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Ethanol recovery process\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe liquid obtained following the recovery of fucoidan and laminarin underwent ethanol recovery using a rotary evaporator (Rotavapor R-300, Buchi, Switzerland), and the reclaimed ethanol was used for alginate extraction. The ethanol-recovered water extracts were observed to contain a variety of polyphenols and pigments.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Laminarin, fucoidan and alginate measurements using high-performance liquid chromatography - refractive index detector (HPLC-RI)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe methodology for quantifying laminarin fucoidan and alginate was refined based on the research conducted by Zhang and Row (2015). In this study, a high-performance liquid chromatography (HPLC) approach was utilized, utilizing an Agilent 1200 LC system (manufactured by Agilent Technologies, located in Santa Clara, California, USA). This HPLC apparatus was outfitted with a refractive index detector and was supported by a guard column (OHpak SB-G 6B, 8 x 50 mm) along with a Shodex OHpak SB-804 HQ carbohydrate column. This column featured 6% cross-linking and dimensions of 8 x 300 mm (length x inner diameter), produced by Shodex, Japan. Preparation of all samples was standardized at a concentration of 2 mg/mL in the chosen running solvent, followed by filtration through 0.45 \u0026micro;m Econo Filters (made of PTFE, supplied by Agilent) for clarity and purity.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Fourier-transform infrared spectroscopy (FT-IR) measurements\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFT-IR spectra of the samples were recorded to identify potential variations in functional groups compared to the standard. The samples were positioned on the surface of a diamond crystal attenuated total reflectance (ATR) accessory (iD7 ATR, Thermo Scientific, Madison, WI, USA), and spectral measurements were conducted with a Fourier transform mid-infrared spectrometer (Nicolet\u0026trade; iS5, Thermo Scientific, Madison, WI, USA). Single-beam spectra were recorded in transmission mode over the range of 4000\u0026ndash;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with 64 scans at a resolution of 0.3 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the differences were documented. Air blank background calibration was conducted prior to each measurement. Each measurement consisted of 64 scans to obtain averaged spectral data. Spectral data acquisition was supervised using the provided OMNIC software version 9.2.98 (Thermo Fisher Scientific Inc., USA)(Rajauria et al., 2023).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of Laminarin/fucoidan\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDPPH was evaluated to showcase the antioxidant activities. Briefly, Seaweed (macroalgae) extracts as samples and ascorbic acid as a positive control were prepared to 1 mg/mL in 0.1 M citrate phosphate buffer with 0.3% of Triton X-100. The samples were prepared in triplicates. Then, 10 \u0026micro;L of a 2 mM methanolic DPPH solution was added to each well. Further, the reaction mixture was incubated at room temperature in the dark for 30 min and read against the blank at 515 nm before and after the reaction with the DPPH solution using a UV-Vis spectrophotometer. The inhibition percentage of DPPH scavenging activity was calculated using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:DPPH\\:radical\\:scavenging\\:activity\\:\\left(\\%\\right)=\\frac{{Abs}_{Blank}-{Abs}_{Sample}}{{Abs}_{Blank}}⨯100$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ewhere \u0026ldquo;Abs\u003csub\u003eBlank\u003c/sub\u003e\u0026rdquo; is the absorbance of the Blank (DPPH solution without sample/standard), \u0026ldquo;Abs\u003csub\u003eSample\u003c/sub\u003e\u0026rdquo; is the absorbance of the test sample (DPPH solution plus test sample/standard).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Sodium alginate characterisation\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Preparation of sodium alginate/Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e hydrogel beads\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e was added to the sodium alginate (SA) solution at a rate of 12.8 g per 100 mL. SA solutions (2 g per 100 mL) were prepared, allowed to fully hydrate by storing them at 4\u0026deg;C for 24 hours, and degassed using ultrasound. Beads were created by slowly dispensing 20 mL of the SA solution into 160 mL of CaCl\u003csub\u003e2\u003c/sub\u003e solution using a 1.2 mm (18\u0026frac12; G) needle. The beads were left in a gelling bath to solidify at room temperature (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) for 30 minutes, then collected by filtration through a plastic mesh and washed three times with deionized water.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Textural properties of the beads\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTextural properties of the beads was evaluated by Lozano-Vazquez et al (2015). The primary textural properties, including hardness, springiness, and cohesiveness of the hydrogel calcium alginate beads, were assessed using a stable micro systems texturometer model TA-XT2i (Texture Technologies Corp., White Plains, NY, USA) fitted with a 30 kg load cell. To ensure reliable test reproducibility, given the particulate nature of the beads, a cylindrical steel probe with a substantial contact area (36 mm in diameter) was employed. Measurements were conducted at room temperature (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) using 30 g of hydrogel calcium alginate beads positioned on a stationary glass plate beneath the probe. Automatic detection of probe-bead contact was performed with a contact force of 0.005 N. The samples underwent 30% compression through two cycles, each at a consistent crosshead velocity of 1.0 mm/s. Textural property values were acquired using the Texture Expert Software for Windows, Version 3.2, integrated with the equipment.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Protein content in residues\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe protein content (%) of the freeze-dried seaweed residues was determined by weighing and then analyzed using a nitrogen analyzer (FP-328, Leco Instrument, Leco Corporation, USA) following the Dumas principle (method 968.06, Official Methods of Analysis of AOAC International, 16th edition, Arlington, VA, USA: AOAC International, 1995) with a conversion factor of 6.25.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Total polyphenol content (TPC)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA 100 \u0026micro;L portion of the extracts was mixed with 2mL 2% (w/v) Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e and allowed to mix for 2 minutes at room temperature. Subsequently, 100 \u0026micro;L of Folin\u0026ndash;Ciocalteu's reagent 1 M was added. The samples were kept in the dark at room temperature for 30 minutes, and the absorbance was measured at 720 nm. A sample blank (containing only solvent without a sample) was included. Gallic acid served as the standard, and the measured absorbance was converted to gallic acid equivalents using a calibration curve constructed with gallic acid. The calibration curve was generated by dissolving gallic acid in the same solvent as the extracts (either methanol or ethyl acetate) within a concentration range of 0 to 0.5 mg mL⁻\u0026sup1;.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEach pre-treatment condition was replicated three times. Subsequently, each sample was analyzed three times to ensure the accuracy and reliability of the results. The statistical analysis of experimental data was conducted using one way analysis of variance (ANOVA), facilitated by the Statistical Package for the Social Sciences (SPSS) software, version 20.0.0 (IBM, U.S.A.). This approach allowed for the identification of significant differences between the treatment groups, with a predefined significance threshold set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data management, including the organization and preliminary analysis, as well as graphical representations of the findings, were handled using Microsoft Excel. For more complex graphical representations, MATLAB (version, company, America) software was employed.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results \u0026 Discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Biorefinery efficiency\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e provides a comparative overview of the purity rates of laminarin, fucoidan, and alginate under different pre-treatment conditions. For laminarin, fresh treatment yielded a laminarin purity of 48.32% in \u003cem\u003eAscophyllum nodosum\u003c/em\u003e, contrasting with a much lower purity of 10.98% in \u003cem\u003eFucus vesiculosus\u003c/em\u003e, indicating inherent differences in their biological composition or structure that affect compound extraction. The dry treatment significantly boosted laminarin purity in \u003cem\u003eAscophyllum nodosum\u003c/em\u003e to 61.46%, while also elevating \u003cem\u003eFucus vesiculosus\u003c/em\u003e' purity to 39.63%. This suggests that drying facilitates the breakdown of cellular barriers, making laminarin more accessible (Kadam et al., 2015). Interestingly, the soaked treatment drastically reduced laminarin purity in \u003cem\u003eA. nodosum\u003c/em\u003e to a mere 0.55%, while \u003cem\u003eF. vesiculosus\u003c/em\u003e, experienced an increase to 38.96%, highlighting a species-specific response that may involve differences in water absorption or laminarin solubilization.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePurity (%) of laminarin, Fucoidan and sodium alginate\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003ePurity Rates(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaminarin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFucoidan\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlginate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFresh Asco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e66.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFresh Fucus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e93.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry Asco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e61.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e68.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry Fucus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoaked Asco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e67.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoaked Fucus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDPPH of laminarin and sodium alginate\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDPPH (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFresh Asco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e49.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFresh Fucus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e47.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry Asco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e62.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry Fucus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.69\u0026thinsp;\u0026plusmn;\u0026thinsp;5.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSocked Asco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSocked Fucus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e24.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe extraction of fucoidan presented a clear advantage with the drying process, where both \u003cem\u003eA. nodosum\u003c/em\u003e and \u003cem\u003eF. vesiculosus\u003c/em\u003e saw significant increases in purity, reaching 43.63% and 39.63%, respectively. This enhancement likely occurs from the dehydration effect altering the seaweed\u0026rsquo;s cell structure in a manner conducive to fucoidan release. Conversely, soaking had a detrimental impact, particularly on Fucus, where purity plummeted to 6.28%, indicating that fucoidan extractability is adversely affected by aqueous conditions, possibly due to degradation or leaching effects.\u003c/p\u003e \u003cp\u003eAlginate extraction offered additional insights into the complexity of pre-treatment effects. Fresh samples showed high alginate purity, especially in Fucus at 93.15%, indicating a more readily extractable state in its natural form, possibly due to higher alginate content or structural differences. The drying process slightly increased alginate purity in \u003cem\u003eAscophyllum nodosum\u003c/em\u003e but decreased it in \u003cem\u003eFucus vesiculosus\u003c/em\u003e, suggesting a delicate balance between moisture content and alginate's extractability or stability. Soaking proved to be less beneficial for alginate extraction, particularly affecting \u003cem\u003eF. vesiculosus\u003c/em\u003e, where purity decreased significantly to 42.11%. This could be attributed to the leaching of alginate into the soaking medium or alterations in its molecular configuration in the presence of excess water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 FT-IR analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe raw FT-IR absorbance spectra of extracted sodium alginate and mixtures of laminarin and fucoidan collected over the wavelength range of 4000-400cm\u003csup\u003e-1\u003c/sup\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e respectively, compared with the standard, Laminarin-S, Fucose-S, Glucan-S and Alginate-acid-s.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe spectra exhibit an increase in absorbance intensity in the O-H stretching vibrations within the 3200\u0026ndash;3600 cm⁻\u0026sup1; range and C-H stretching vibrations around 2920 cm⁻\u0026sup1; (Garza-Cervantes et al., 2019). This enhancement was particularly pronounced in samples subjected to soaking treatments, such as Soaked \u003cem\u003eA. nodosum\u003c/em\u003e and Soaked \u003cem\u003eF. vesiculosus\u003c/em\u003e, indicating a potential increase in hydroxyl and alkyl chain contents across both laminarin, fucoidan, and alginate samples. The elevated absorbance suggests that soaking methods may modify the water-binding capacity and hydrophobic interactions within these polysaccharides, enhancing their extraction yields.\u003c/p\u003e \u003cp\u003eThe analysis revealed a marked increase in absorbance at 1620 cm⁻\u0026sup1; and 1420 cm⁻\u0026sup1;, indicative of carboxylate functional groups, across all processed samples (Frota et al., 2024). The increase was most significant in alginate samples undergoing soaking treatment, highlighting that the soaking process facilitates the exposure or increases the content of carboxylate groups, crucial for the functionality and solubility of alginate.\u003c/p\u003e \u003cp\u003eThe spectral analysis within the 1320\u0026thinsp;\u0026minus;\u0026thinsp;1260 cm⁻\u0026sup1; and 1080\u0026thinsp;\u0026minus;\u0026thinsp;1030 cm⁻\u0026sup1; regions, corresponding to C-O-C and C-O stretching vibrations, showed that processed samples displayed higher absorbance intensity than the standard references for laminarin, fucoidan, and alginate (Khalafu et al., 2017). This increase, especially notable in soaked samples, implies that soaking treatments may preserve or enhance the polysaccharide structures.\u003c/p\u003e \u003cp\u003eSpecifically, for fucoidan, the absorbance intensities at wavelengths indicative of sulfate ester groups (approximately 1250 cm⁻\u0026sup1; and 820 cm⁻\u0026sup1;) were higher in samples processed through soaking treatments (Mohd Fauziee et al., 2021). This observation suggests an increase in sulfation levels or better preservation of sulfate ester groups, highlighting the specificity of soaking effects on fucoidan's structural components.\u003c/p\u003e \u003cp\u003eThe integrated spectroscopic analysis underscores the nuanced impact of soaking treatments on the extraction efficiency and structural preservation of laminarin, fucoidan, and alginate. The enhanced absorbance intensity in key functional groups across all polysaccharides suggests that soaking treatments can significantly improve yield and quality by altering the hydrophilic and hydrophobic balance and exposing more functional groups essential for their biological and physicochemical properties.\u003c/p\u003e \u003cp\u003eSoaking treatments, particularly those applied to Socked \u003cem\u003eA. nodosum\u003c/em\u003e and Socked \u003cem\u003eF. vesiculosus\u003c/em\u003e, samples, have demonstrated superior performance in terms of extraction efficiency, outperforming fresh and dried samples. This indicates that soaking not only enhances the solubility of these polysaccharides but may also facilitate the optimization of the extraction process and enhance product quality through better exposure and preservation of essential functional groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Antioxidant activities of fucoidan and laminarin mixtures (DPPH)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe antioxidant capacities of fucoidan and laminarin mixtures were evaluated using the DPPH assay, which revealed significant effects of the pre-treatment on antioxidant activity. For the mixtures extracted from \u003cem\u003eA. nodosum\u003c/em\u003e, fresh samples showed an antioxidant activity of 49.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79%. This significantly increased to 62.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.556% after drying (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting that drying may help concentrate or alter the extraction efficiency of antioxidant compounds. However, the activity markedly decreased in soaked samples to 30.14\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61%, likely due to the leaching of antioxidant components or degradation of active compounds in aqueous conditions. According to Siriwardhana et al.(2003) and Liu and Ng (2000), a strong correlation (r\u0026thinsp;=\u0026thinsp;0.971) was found between the DPPH radical-scavenging activities and total polyphenolic content, which may also explain dry samples have high antioxidant potential compounds that less antioxidant compounds leak with phenolic compounds. However, there are higher total polyphenolics content in the soaked samples\u0026rsquo; water extracts with more antioxidant potential compounds leaking.\u003c/p\u003e \u003cp\u003eConversely, the mixtures extracted from \u003cem\u003eF\u003c/em\u003e. \u003cem\u003evesiculosu\u003c/em\u003e displayed an antioxidant activity of 47.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.162% in fresh samples, which significantly decreased after drying to 26.69\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1999%, and slightly further decreased in soaked conditions to 24.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.968% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which is consistent with the findings of Jim\u0026eacute;nez-Escrig et al (2001). This significant reduction could be associated with structural or compositional differences between the two types of seaweed, which may influence the stability and extractability of antioxidant compounds under different treatment conditions.\u003c/p\u003e \u003cp\u003eThese results indicate that the method of pre-treatment significantly impacts the antioxidant potential of fucoidan and laminarin mixtures. These variations highlight the necessity of optimizing processing conditions tailored to each seaweed type to maximize the recovery of beneficial antioxidants, crucial for potential applications in nutraceuticals and functional foods.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Properties of alginate (calcium alginate-modified beads)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the geometrical and textural characteristics of calcium alginate-modified beads with 3 different treatments of \u003cem\u003eAscophyllum nodosum\u003c/em\u003e and \u003cem\u003eFucus vesiculosus\u003c/em\u003e. Comparing the alginate properties of \u003cem\u003eA. nodosum\u003c/em\u003e obtained through different treatments, it can be seen that three treatments exert a significant impact on three critical textural parameters: hardness, viscosity and chewiness (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, parameters such as adhesiveness, elasticity, cohesiveness and resilience did not exhibit statistically significant alterations (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe geometrical and textural characteristics of calcium alginate-modified beads\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHardness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdhesiveness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSpringiness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCohesiveness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGumminess\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eChewiness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eResilience\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFresh Asco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e170.726\u0026thinsp;\u0026plusmn;\u0026thinsp;65.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-1.821\u0026thinsp;\u0026plusmn;\u0026thinsp;0.611\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.736\u0026thinsp;\u0026plusmn;\u0026thinsp;0.042\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e126.813\u0026thinsp;\u0026plusmn;\u0026thinsp;51.808\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e95.047\u0026thinsp;\u0026plusmn;\u0026thinsp;43.056\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.411\u0026thinsp;\u0026plusmn;\u0026thinsp;0.052\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFresh Fucus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e276.081\u0026thinsp;\u0026plusmn;\u0026thinsp;25.132\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.374\u0026thinsp;\u0026plusmn;\u0026thinsp;0.691\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.042\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.714\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e197.31\u0026thinsp;\u0026plusmn;\u0026thinsp;19.309\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e143.986\u0026thinsp;\u0026plusmn;\u0026thinsp;15.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.437\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry Asco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e192.318\u0026thinsp;\u0026plusmn;\u0026thinsp;15.928\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.961\u0026thinsp;\u0026plusmn;\u0026thinsp;0.881\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.788\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.745\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e143.198\u0026thinsp;\u0026plusmn;\u0026thinsp;10.937\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e112.907\u0026thinsp;\u0026plusmn;\u0026thinsp;10.467\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.435\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry Fucus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e266.555\u0026thinsp;\u0026plusmn;\u0026thinsp;9.152\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.816\u0026thinsp;\u0026plusmn;\u0026thinsp;3.128\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.779\u0026thinsp;\u0026plusmn;\u0026thinsp;0.044\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.065\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e197.642\u0026thinsp;\u0026plusmn;\u0026thinsp;23.386\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e154.555\u0026thinsp;\u0026plusmn;\u0026thinsp;26.232\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.464\u0026thinsp;\u0026plusmn;\u0026thinsp;0.068\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSocked Asco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e191.501\u0026thinsp;\u0026plusmn;\u0026thinsp;22.274\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-4.602\u0026thinsp;\u0026plusmn;\u0026thinsp;1.617\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.795\u0026thinsp;\u0026plusmn;\u0026thinsp;0.034\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.767\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e146.752\u0026thinsp;\u0026plusmn;\u0026thinsp;16.072\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e117.046\u0026thinsp;\u0026plusmn;\u0026thinsp;17.434\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.395\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSocked Fucus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e228.596\u0026thinsp;\u0026plusmn;\u0026thinsp;8.182\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-33.184\u0026thinsp;\u0026plusmn;\u0026thinsp;10.933\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.771\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.749\u0026thinsp;\u0026plusmn;\u0026thinsp;0.042\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e171.245\u0026thinsp;\u0026plusmn;\u0026thinsp;12.969\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e132.088\u0026thinsp;\u0026plusmn;\u0026thinsp;10.482\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.043\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eNote: Data in the same column with the same letter are not significantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05); all data are the means from 3 replicates.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe pronounced disparities in hardness and viscosity imply that the treatments can modulate the density and strength of cross-linkages amongst alginate molecules, subsequently altering the structural integrity and mechanical attributes of the modified beads. This observation is congruent with prior studies, which have posited that pretreatment conditions, including drying and soaking, can influence the physical and chemical properties of alginates\u0026mdash;namely, their molecular weight distribution and solubility\u0026mdash;thereby affecting their functional properties, such as gel formation capacity and adhesiveness (McHugh, 1987). The marked variation in chewiness further emphasizes the significant role of treatments in modulating the mechanical performance of the modified beads, an aspect crucial for their potential functional applications within the realms of food processing and biomedical endeavours.\u003c/p\u003e \u003cp\u003eThe lack of significant differences in adhesiveness, elasticity, cohesiveness, and resilience may reflect the insensitivity of these textural characteristics to variations in pretreatment conditions, or possibly, limitations inherent to the experimental design and statistical analysis employed. For example, an increase in the sample size or the adoption of more sensitive measurement techniques might unveil subtle variations in these attributes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Protein/Fibre\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the protein content across different samples and treatments. An ANOVA test was conducted to compare the protein content (%) across different treatment methods for each freeze-dried seaweed residue and raw materials. The result indicated a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in protein content across different treatments, which are critical for optimizing seaweed bio-refinery processes.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe residue with dry treatment exhibited the highest protein contents among the tested conditions, particularly notable in \u003cem\u003eAscophyllum nodosum\u003c/em\u003e (Dry Asco) with an average protein content of 16.66%, suggesting a concentration effect due to moisture removal, beneficial for applications requiring high protein yields. Conversely, soaked treatments resulted in reduced protein levels, likely due to the acid used which causes protein molecules to leach (Kadam et al., 2017).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 TPC\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWater extracts after extracting the mixture of laminarin/fucoidan were collected. The comparative study demonstrated significant differences in TPC across the pre-treatments for each type of seaweed, with soaked samples consistently exhibiting the highest phenolic content across both seaweed types. Specifically, Water extracts of \u003cem\u003eFucus vesiculosus\u003c/em\u003e treated through soaking showed a remarkable increase in TPC concentration, indicating an enhanced extraction of phenolic compounds due to the pre-treatment process. The increase could be a result of acidification disintegrating cell walls of phenolic compounds, which helps facilitate phenolic compounds the solubilization and diffusion during the extraction (Charoensiddhi et al., 2015). This is in stark contrast to the dry treatment, which generally resulted in the lowest TPC values among the three methods evaluated. This result is consistent with the previous reports by Gupta et al., (2011). For \u003cem\u003eAscophyllum nodosum\u003c/em\u003e, while the difference in TPC between treatments was less pronounced than in \u003cem\u003eFucus vesiculosus\u003c/em\u003e, the soaked treatment still emerged as the most effective method for maximizing phenolic content extraction. The fresh treatment showed intermediate results, and similar to \u003cem\u003eFucus vesiculosus\u003c/em\u003e, the dry treatment yielded the lowest TPC values.\u003c/p\u003e \u003cp\u003eThe water extracts of \u003cem\u003eFucus vesiculosus\u003c/em\u003e showed a considerable variation in TPC among treatments. In contrast, \u003cem\u003eAscophyllum nodosum\u003c/em\u003e's TPC was not significantly affected by the treatments.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe comprehensive analysis of different pre-treatment methods in this study revealed the complicate impacts these methods have on the extraction efficiencies and qualities of laminarin, fucoidan, and alginate from Ascophyllum nodosum and Fucus vesiculosus. The research demonstrates that drying pre-treatments significantly enhances the extraction of laminarin and fucoidan by facilitating the breakdown of cellular barriers, thereby making these bioactive compounds more accessible. Conversely, soaking pre-treatments exhibit species-specific responses, which affect the purity levels of both laminarin and alginate differently, indicating a complex interplay between seaweed species, water absorption, and polysaccharide solubilization dynamics. Additionally, the FT-IR analysis provides a molecular perspective, demonstrating how various pre-treatments can modify the structural integrity and functional group exposure of the target polysaccharides, influencing their potential bioactive applications. This comprehensive examination of pre-treatment effects not only advances our understanding of seaweed bio-refinery processes but also highlights the importance of selecting appropriate pre-treatment strategies to optimize the yield and bioactivity of extracted compounds, laying a foundation for future innovations in the sustainable utilization of marine resources for nutraceutical, pharmaceutical, and agricultural applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCRedit authorship contribution statement\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYiting Han:\u003c/strong\u003e Conceptualization, Methodology, Formal analysis, Data curation, Writing - original draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXinyu Tan:\u0026nbsp;\u003c/strong\u003eMethodology, Data curation, Writing - original draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStephen Fitzpatrick:\u003c/strong\u003e Methodology, Data curation, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHenry Lyons:\u003c/strong\u003e Methodology, Data curation, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXianglu Zhu:\u003c/strong\u003e Conceptualization, Project administration, Formal analysis, Data curation, Writing - review \u0026amp; editing, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrijesh K Tiwari:\u003c/strong\u003e Conceptualization, Project administration, Funding acquisition, Formal analysis, Data curation, Writing - review \u0026amp; editing, Supervision.\u003c/p\u003e\n\u003cp\u003eDeclaration of Competing Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnisha, G.S., Padmakumari, S., Patel, A.K., Pandey, A., Singhania, R.R., 2022. 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Algal Research 75, 103277. https://doi.org/10.1016/j.algal.2023.103277\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ascophyllum nodosum, Fucus vesiculosus, Biorefinery, Laminarin, Fucoidan, Alginate","lastPublishedDoi":"10.21203/rs.3.rs-5696543/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5696543/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis paper investigates the impact of different pre-treatments on the bio-refinery processes of wild-harvested Irish brown seaweeds, specifically \u003cem\u003eAscophyllum nodosum\u003c/em\u003e and \u003cem\u003eFucus vesiculosus\u003c/em\u003e. Employing a combination of mechanical and chemical methodologies, including drying, soaking, and the application of specific reagents, we aimed to optimize the extraction of valuable polysaccharides such as laminarin, fucoidan, and alginate, alongside protein recovery. The research highlighted the significant influence of pre-treatment methods on extraction efficiencies and polysaccharide purity, indicating that drying is beneficial for improving the purity and yield of laminarin and fucoidan, with laminarin purity as high as 61.46% in \u003cem\u003eAscophyllum nodosum\u003c/em\u003e. The study also demonstrates a complex interplay in alginate extraction across different treatments, with fresh treatments achieving up to 93.15% purity in \u003cem\u003eFucus vesiculosus.\u003c/em\u003e FT-IR provided insight into structural alterations and functional group exposure of extracted polysaccharides, indicating the potential of pre-treatment strategies in enhancing the yield and quality of bioactive compounds. These findings advance our understanding of seaweed bio-refinery processes and underscore the importance of pre-treatment selection in maximizing the sustainable utilization of marine resources for pharmaceutical, nutraceutical, and agricultural applications.\u003c/p\u003e","manuscriptTitle":"Impact of pre-treatment strategies for enhance conversion of Irish Brown Seaweed into high value ingredients using a biorefinery approach.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-17 10:41:37","doi":"10.21203/rs.3.rs-5696543/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"16a6ae28-1528-49f0-ab6b-5628e9aa1df7","owner":[],"postedDate":"January 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-22T14:21:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-17 10:41:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5696543","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5696543","identity":"rs-5696543","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}