Evaluation of phlorotannin bioaccessibility and antioxidant capacity stability of a safe and sustainable brown seaweed extract: role of age-related digestion changes and matrix composition

Evaluation of phlorotannin bioaccessibility and antioxidant capacity stability of a safe and sustainable brown seaweed extract: role of age-related digestion changes and matrix composition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of phlorotannin bioaccessibility and antioxidant capacity stability of a safe and sustainable brown seaweed extract: role of age-related digestion changes and matrix composition Migdalia Arazo-Rusindo, Gonzalo Rivera-Andrades, Layla Simón, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7427244/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Food Production, Processing and Nutrition → Version 1 posted You are reading this latest preprint version Abstract Addressing age-related health challenges through improved nutrition is increasingly important as the global population ages. Concurrently, sustainable food systems are focusing on low-impact ingredients, such as brown seaweed, which require minimal resources and offer promising health benefits. However, concerns about heavy metal accumulation in brown seaweed highlight the challenges associated with its safe use. After confirming the safety of a sustainable Durvillaea incurvata extract, containing phlorotannins from trimers to octamers and mannitol, through an 80% reduction in heavy metals, this study evaluated the bioaccessibility of phlorotannins and the stability of antioxidant capacity during in vitro gastrointestinal digestion in both standard and older adult models. It also compared these results with two commercially available seaweed products: micronized capsules and rehydrated salad. Crude extract exhibited the highest phlorotannin level before digestion for both Folin-Ciocalteu (22.5 mg PhE/g dw) and DMBA (0.8 mg PhE/g dw) assays. Phlorotannin bioaccessibility indices reached 1005% and 332% in the standard and older adult models, respectively, when total soluble polyphenol values were used for measurement. The DMBA assay confirmed an increase in phlorotannin content after digestion, likely due to the release of compounds previously bound to the food matrix; however, the differences observed between the two digestion models did not exhibit the same magnitude of change. The antioxidant capacity (DPPH assay) was highest in the micronized seaweed; however, it decreased after digestion. Conversely, the digested crude extract retained its antioxidant capacity under older adult conditions. These results support the use of brown seaweed extracts as safe and effective ingredients in foods for older adults. seaweed phlorotannin antioxidant capacity bioaccessibility older adults Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The global population is aging rapidly, with estimates suggesting that by 2050, individuals aged 60 and above will constitute nearly 22% of the worldwide population. This demographic shift brings with it significant health challenges, particularly for older adults, who are prone to various chronic diseases such as cardiovascular diseases, diabetes, and osteoporosis (WHO, 2024). Among the less visible but equally impactful health concerns for older populations are gastrointestinal issues, which can lead to malabsorption and nutritional deficiencies (Arazo-Rusindo et al., 2022 ; Bustos-Arriagada et al., 2024 ). Digestive problems, including reduced stomach acid production, slower gut motility, and altered gut microbiota composition, are common in older individuals. They can significantly impair the absorption of essential nutrients, leading to deficiencies in vitamins, minerals, and proteins (Dumic et al., 2019 ; Menard et al., 2023 ). These deficiencies can exacerbate age-related frailties, increasing the risk of muscle wasting (sarcopenia), bone demineralization, and immune system decline (Gallo et al., 2024 ). Given these health concerns, it has become increasingly important to design tailored food products that specifically meet the nutritional needs of older adults. Customized nutrition for the elderly involves addressing not only the macronutrient and micronutrient requirements but also integrating bioactive compounds, such as polyphenols, into food matrices that can help mitigate the onset of chronic diseases. Incorporating polyphenols into functional foods for older adults presents a viable approach to address the digestive and chronic disease-related challenges of this demographic, particularly given the increasing evidence on the role of these compounds in improving intestinal health through various mechanisms of action (Espín et al., 2017 ). However, ensuring the post-digestion bioavailability of these compounds is crucial. Phlorotannins, a specific class of polyphenols found in brown seaweed, have emerged as up-and-coming candidates for functional food and pharmaceutical applications due to their antioxidant (Chen et al., 2023 ; Erpel et al., 2021 ; Rajauria et al., 2013 ), antidiabetic (Gheda et al., 2021 ), anti-inflammatory (Besednova et al., 2022 ; S. Lee et al., 2019 ) and anti-carcinogenic properties (Catarino et al., 2020 ; Cotas et al., 2020 ), although clinical trials with these compounds are still scarce (Keleszade et al., 2021 ). In parallel, brown seaweeds are recognized as low-impact food ingredients due to their rapid growth, minimal freshwater and fertilizer requirements, and their capacity to absorb carbon dioxide during cultivation. These characteristics establish seaweeds as promising sustainable resources within the framework of climate change and resource-limited food systems (Cherry et al., 2019 ). In this sense, different studies have evaluated sustainable extraction processes, such as ultrasound-assisted extraction, to obtain natural extracts from seaweed, which contain phloroglucinol polymers and mannitol (Muñoz-Molina et al., 2025 ; Rivera-Tovar et al., 2025 ). Nevertheless, the use of phlorotannin-rich seaweed products remains limited (Pacheco et al., 2023 ), partly due to concerns regarding seaweeds' potential to accumulate toxic heavy metals (Erpel et al., 2020 , 2021 ). Consequently, it is essential to ensure that seaweed-based ingredients contain safe levels of these harmful compounds before including them in food products at non-cytotoxic concentrations. Bioaccessibility and bioavailability are key aspects to consider when using polyphenols as dietary ingredients, given the extent of their metabolic fate after intake, ultimately resulting in final bioactive metabolites (Cortés-Martín et al., 2020 ). However, little information exists on how phlorotannins transform after digestion and their resulting biological activity (Corona et al., 2017 ; Subbiah et al., 2024 ). Moreover, recent studies have shown that the age-related changes in the digestion process can decrease the bioavailability of phenolic compounds, as observed after in vitro digestion of apples or chia seeds (Hernández-Olivas et al., 2023 ; Shang et al., 2022 ). Furthermore, the food matrix plays a crucial role in regulating the release and absorption of these bioactive compounds during the digestion process (Qazi et al., 2025 ; Y. Zhang et al., 2024 ). Meeting the nutritional needs of the aging population requires a comprehensive approach that combines delivering essential nutrients with including bioactive compounds, such as phlorotannins, while also evaluating their bioaccessibility and identifying factors that affect their absorption in the intestine. This study investigates the bioaccessibility of phlorotannins and the stability of their antioxidant capacity after simulated gastrointestinal digestion, considering the physiological changes associated with aging. It evaluates three Durvillaea incurvata -based matrices produced through various processing methods: a fresh seaweed salad (SS), micronized seaweed (MS), and an innovative seaweed extract (CE) containing phlorotannins (trimers to octamers of phloroglucinol) and mannitol, specifically designed to address issues of sustainability, efficacy, and safety. MATERIALS AND METHODS Two commercially available Durvillaea incurvata foods (MS and SS) were compared with a CE obtained through a sustainable and scalable process to evaluate their potential as phlorotannin oral delivery systems. The CE had been previously found to contain mannitol and phlorotannins with structural features associated with anti-inflammatory activity (Rivera-Tovar et al., 2025 ). Safety was first assessed by determining the heavy metal content of both raw material and CE, followed by a cytotoxicity assay to establish a safe dose for use. The proximate composition of CE, MS, and SS was also analyzed. After completing safety assessment and preliminary chemical characterization, the three products were subjected to in vitro digestion using two INFOGEST 2.0 protocols: the standard model (Brodkorb et al., 2019 ) and an adaptation representing older adults (Menard et al., 2023 ), to evaluate phlorotannin bioaccessibility and antioxidant stability (Fig. 1 ). 2.1. Materials 2.1.1. Durvillaea Incurvata samples Durvillaea incurvata fronds used for the extract were collected from the coast of Concepción, Chile (36°32′55″S 72°56′10″W) during the fall of 2023. Seaweed Place SPA, Chile, kindly provided the MS and SS. 2.1.2. Chemicals and analytic reagents Distilled water, methanol (99.9%, HPLC grade), acetone (99.8%, HPLC grade), and acetic acid (99.0%), sodium carbonate (Na 2 CO 3 ), dipotassium phosphate (K 2 HPO 4 ), potassium dihydrogen phosphate (KH 2 PO 4 ), ammonium carbonate ((NH 4 )2CO 3 ), calcium chloride dihydrate (CaCl 2 ·2H 2 O), magnesium chloride hexahydrate (MgCl 2 · 6H 2 O), potassium chloride (KCl), potassium dihydrogen phosphate (KH 2 PO 4 ), sodium chloride (NaCl) and sodium hydroxide (NaOH), hydrochloric acid 37% (HCl), α-amylase from porcine pancreas (1.25×10 − 6 kat/mL), pepsin from porcine gastric mucosa (3.33 ×10 − 5 kat/mL), pancreatin from porcine pancreas (as trypsin activity 1.67×10 − 6 kat/mL) and porcine bile salts (7.07 g/100 mL), were purchased from Merck LifeScience S.L.U. (Santiago, Chile). The Folin–Ciocalteu reagent, DPPH (1,1-Diphenyl-2-picryl-hydrazil), fluorescein sodium salt, AAPH (2,2´-azobis-2-methylpropionamidine dihydrochloride), standards of phloroglucinol (99%) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Sigma-Aldrich (Steinem, Germany). 2.2. Methods 2.2.1. Obtaining a sustainable crude extract (CE) from Durvillaea incurvata . The Durvillaea incurvata CE was obtained using ultrasound-assisted extraction (USAE). The fronds were chopped, freeze-dried, ground, and sieved to obtain particles smaller than 710 µm. A 20 g sample was mixed with 200 mL of 32.5% ethanol/water solution (Rivera-Tovar et al., 2025 ). USAE was performed with an ultrasonic probe (BIOBASE, China) at 20 kHz, 40% power (600 W), 30°C, and a pulse cycle of 5/15 seconds. The mixture was centrifuged at 2490×g for 5 minutes, and the liquid phase was concentrated by rotary evaporation at 40°C for 90 minutes. The resulting extract was freeze-dried at -80°C for 24 hours to produce a stable powder. The CE used in this study was previously characterized in detail by our group (Rivera-Tovar et al., 2025 ). In that work, FTIR analysis confirmed the presence of functional groups associated with both polyphenols and mannitol, while HPLC-IR quantification indicated a mannitol content of 259 ± 24 mg/g dried extract. Additionally, UHPLC-QToF-MS enabled the tentative identification of phlorotannins ranging from trimers to octamers, including carmalols, fucols, and eckols. 2.2.2. Heavy metals analysis To evaluate the safety of both the dried seaweed and its CE, concentrations of arsenic (As), cadmium (Cd), lead (Pb), mercury (Hg), and tin (Sn) were quantified following the methodology outlined by (Erpel et al., 2021 ). The quantification employed two complementary analytical techniques: atomic absorption spectroscopy (AAS) for As and Hg and inductively coupled plasma atomic emission spectroscopy (ICP-AES) for Cd, Pb, and Sn. Analyses of the dried seaweed samples adhered to the standardized procedures TMECC 04.14 and 04.12-B (Thompson et al., 2001 ). For the CE samples, evaluations followed the ME-12 protocol (Superintendencia de Servicios Sanitarios, 2007 ), supplemented by Standard Methods 3030C and E, and 3120B (APHA et al., 1999). All results were expressed in milligrams of metal per kilogram of dry weight (mg/kg DW), with each analysis conducted in duplicate to ensure analytical accuracy. 2.2.3. Cytotoxicity assay The effect of CE on cell viability was determined using the MTS assay as described previously (Simón et al., 2024 ). Briefly, 3 × 10^3 HEK293T and HT29 cells were seeded in 96-well plates. After an initial 24-hour period in culture, cells were treated with increasing concentrations of CE (0–4 g/100 mL) for 48 hours. Viability was measured according to the manufacturer’s instructions (Cat. #G3580, Promega, Madison, WI). 2.2.4. Proximate analysis The proximate composition of Durvillaea incurvata and its three based products (CE, MS, and SS) was assessed. Moisture content was measured using the thermogravimetric method at 105°C in an oven, by the Chilean standard NCh 841.Of78 (INN, 1978). Protein content was determined using the Kjeldahl method, as specified in the Chilean standard NCh 2748. Of.2019 (INN, 2019), with a nitrogen-to-protein conversion factor of 6.25. Oil content was measured through ether extraction in a Soxhlet apparatus according to AOAC Official Methods of Analysis, method 920.39 (AOAC International, 2016 ). Ash content was obtained by incineration at 550°C in a muffle furnace, following AOAC Official Method 942.05 (AOAC International, 2012 ). Total, soluble, and insoluble dietary fiber were analyzed using the enzymatic-gravimetric method, as outlined in AOAC Official Method 991.43 (AOAC International., 2016). Available carbohydrates were calculated by difference based on AOAC Official Method 986.25 (AOAC International., 2012). 2.2.5. Determination of phlorotannin content The phlorotannin content of both undigested and digested samples was determined by evaluating their total soluble polyphenol content (TSPC) and total phlorotannin content (PhT). TSPC was measured using the method of (Singleton & Rossi, 1965 ). Briefly, 20 µL of the standard extracts or extraction solution (blank) were sequentially mixed with 10 µL of Folin-Ciocalteu reagent (diluted 1:2 with deionized water) and 150 µL of deionized water. The solution was homogenized, and 20 µL of a 10% Na 2 CO 3 solution (w/v) was added. The mixture was left to react in the dark at room temperature for one hour. Absorbance readings were recorded at 750 nm using a multi-mode microplate reader (Biotek Instruments, Winooski, VT, USA) and compared to a phloroglucinol calibration curve (15–150 mg/L). Each analysis was performed in triplicate, and the results were expressed as mg of phloroglucinol equivalents (mg PhE) per g of dry sample. PhT was determined using the DMBA method according to the protocol developed by (Rivera-Tovar et al., 2025 ). Samples were reconstituted (20 mg in 10 mL of water) and analyzed in duplicate. The reaction involved shaking for 30 minutes, followed by incubation at 25°C in the dark for 60 minutes. Absorbance was measured at 515 nm using a 96-well microplate reader (Biotek Instruments, Winooski, VT, USA). Values were interpolated from a phloroglucinol calibration curve (1–40 mg/L) and expressed as milligrams of phloroglucinol equivalents (mg PhE) per g of dry sample. 2.2.6. Determination of the antioxidant capacity Antioxidant capacity was assessed using two complementary assays: a modified version of the 1,1-Diphenyl-2-picryl-hydrazil scavenging capacity (DPPH) method and an adjusted version of the Oxygen Radical Antioxidant Capacity (ORAC) method. DPPH assay was developed as follows: methanolic extracts or Trolox standards (100 µL) were mixed with 100 µL of DPPH solution (0.048 mg/mL) in 96-well plates. After 30 min of incubation in the dark at room temperature, absorbance was measured at 520 nm (Biotek, USA) (Brand-Williams et al., 1995). Blanks were included to correct for sample color. Results were calculated from a Trolox standard curve (1–32 µM) and expressed as µmol Trolox equivalents per gram of dry sample. Analyses were performed in triplicate. The ORAC microassay was performed as described by (Ou et al., 2001 ), with slight adjustments. In a 96-well microplate, 150 µL of a solution containing 96 nM of fluorescein in a 75 mM phosphate buffer (pH 7.4) was combined with 25 µL of either extract or a diluted Trolox standard. The plate was incubated for 30 min at 37°C. The reaction was initiated by adding 50 µL of a 79 mM AAPH solution prepared in the phosphate buffer. Fluorescence readings were taken every minute for 2 hours at 37°C using a multi-mode microplate reader (Biotek Instruments, Winooski, VT, USA), with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The area under the curve (AUC) for samples, standards, and phosphate buffer blanks (AUC blank ) was determined using Gen5 Data Analysis Software (Biotek Instruments). The net AUC values for both samples and standards were calculated based on [Eq. 2]: \(\:\text{N}\text{e}\text{t}\:\text{A}\text{U}\text{C}={\left[AUC\right]}_{sample\:or\:Std}-{\left[AUC\right]}_{blank}\) [Eq. 2] The ORAC values of the samples were calculated by interpolating from the standard curve of Net AUC Std vs. Trolox concentrations (1–32 µM). Results were reported as µmol of Trolox equivalents (µmol T eq) per g of dry sample. All analyses were conducted in triplicate. 2.2.7. In vitro gastrointestinal digestion models CE, MS, and SS samples were digested using an in vitro gastrointestinal system based on the INFOGEST 2.0 protocol under standard conditions (Brodkorb et al., 2019 ) and an adapted version that simulated the physiological conditions of older adults (Menard et al., 2023 ). The digestion process consisted of three phases: oral, gastric, and intestinal, utilizing simulated salivary (SSF), gastric (SGF), and intestinal (SIF) fluids. Fresh SSF, SGF, and SIF ionic electrolyte stock solutions were prepared before the experiments (Supplementary Material S2). After the intestinal phase, the samples were centrifuged at 2490 g for 5 minutes at 4°C to inactivate the enzymes. A control experiment was also conducted without the food matrix to assess the effect of the chemical environment on the response. The in vitro bioaccessibility of phlorotannin and antioxidant stability of the liquid fraction (supernatant) of each sample were estimated based on the recovery index (%) (Eq. 1). \(\:\text{R}\text{e}\text{c}\text{o}\text{v}\text{e}\text{r}\text{y}\:\text{I}\text{n}\text{d}\text{e}\text{x}\:\left(\text{%}\right)\frac{{PC}_{digested\:sample}\:or\:{AA}_{digested\:sample}}{{PC}_{undigested\:sample}\:or\:{AA}_{undigested\:sample}}\text{x}100\) [Eq. 1] Regardless of the digestion model used, the PC digested sample corresponds to the TSPC or PhT, and the AA digested sample corresponds to the antioxidant capacity values of the three seaweed-based matrices after digestion, respectively. Meanwhile, PC undigested and AA undigested samples refer to the TSPC, PhT, and the samples' antioxidant capacity values before in vitro gastrointestinal digestion, respectively. 2.2.8. Statistical analysis Results for viability, TSPC, PhT, radical scavenging capacity (DPPH), and oxygen radical antioxidant capacity (ORAC) were presented as means ± SD of three replicates per sample. Statgraphics Centurion XVIII Software version 18.1.12 (Stat-Point Technologies Inc., Warrenton, VA, USA) was used for the Analysis of Variance (ANOVA) and Fisher’s least significant difference (LSD) test for multiple sample comparisons, with a 95% confidence level ( p < 0.05). Shapiro–Wilk and Levene tests were used to evaluate normality and homoscedasticity, respectively. RESULTS AND DISCUSSION 3.1. Heavy metal content Due to concerns about toxic metal concentrations in seaweeds (Prashant et al., 2025 ), which may counteract their benefits, levels in crude D. incurvata and its CE were measured and compared with authorized values from Chinese and French regulations. It is worth noting that, to date, significant differences and a lack of harmonized international standards or guidelines specifically addressing the food safety of seaweed production persist (Guo et al., 2023 ). In China, maximum levels of inorganic arsenic and lead in seaweed and its food products were established by the National Food Safety Standard on Maximum Levels of Contaminants in Foodstuffs (GB 2762 − 2022) (NHCPRC & SAMR), 2023). For its part, the European Union (EU) has developed specific standards for seaweed-based foodstuffs, setting maximum permissible levels for contaminants such as I, As, Pb, and Cd in food supplements containing between 80% and 100% of dried seaweed (EU, 2018). On the other hand, France was the first European country to establish complete regulation for the maximum levels of heavy metals (inorganic As, Cd, Pb, Hg, and Sn) in seaweed and derived foods (ANSES, 2020). In this sense, our results demonstrate the superior safety of CE over raw seaweed ( D. incurvata ), which aligns with international efforts to regulate seaweed-derived food products. In particular, Cd content in the raw seaweed (7.6 mg/kg DW) exceeded European and French limits (3.0 mg/kg and 0.5 mg/kg, respectively), whereas it was reduced to below 0.2 mg/kg DW in CE. Similarly, Pb levels decreased from 1.1 to 0.9 mg/kg DW, remaining within acceptable limits for seaweed products. Hg levels in both matrices were well below regulatory limits. Overall, CE revealed a significant reduction of more than 80% in the concentration of heavy metals after the USAE process, with values below the maximum limits recommended by French law. These results align with previous research, which has successfully controlled the toxicity potential of D. incurvata by applying selective separation techniques, achieving reductions of at least 70% (Erpel et al., 2021 ). Furthermore, it is important to emphasize that reducing heavy metals is crucial not only for toxicological reasons but also to ensure the safe absorption of bioactive compounds, as the presence of these metals can interfere with the bioavailability of polyphenols (Omoarelojie & van Staden, 2024 ; Toth & Pavia, 2000 ). Recent studies have confirmed that polyphenols, such as phlorotannins, can bind to heavy metals, forming less bioavailable complexes (Connan & Stengel, 2011 ; R. Zhang et al., 2022 ). Thus, efficient removal of these metals in the CE is expected to allow these compounds to maintain their functionality during in vitro digestion. 3.2. Sub-cytotoxic concentration Due to the uncharacterized cytotoxic potential of this novel CE, it was necessary to verify that it does not impact cell viability. An MTS assay was performed to determine the effect of CE on the human kidney non-tumoral HEK293T and human colon cancer HT29 cell lines. The concentration used for the digestion assay (1 g CE / 100 mL) was sub-cytotoxic for both cell lines (Fig. 2 ), thereby demonstrating the safety of the CE. A higher concentration (4 g CE/100 mL) was only toxic to the colon cancer cell line, highlighting the potential antitumoral effect of this CE. 3.3 Changes in phlorotannin levels of seaweed matrices during digestion in both normal and older adult conditions. Once the safety of the CE was established, in terms of heavy metal levels and effects on cell viability, the proximate composition of samples was determined (Table 1 ). The macronutrient composition of seaweed fronds was similar to that previously reported by other authors, confirming the nutritional value of this sustainable resource (Burgos-Díaz et al., 2022 ). Durvillaea incurvata is an excellent source of total fibe r ( ~ 50.0%), making it a promising food for preventing several non-communicable diseases (Parada et al., 2019 ). Interestingly, the proportion of available carbohydrates and soluble fiber found in this research was higher (5-fold and 2-fold, respectively) than the values previously reported for the same specie (Burgos-Díaz et al., 2022 ). This can be explained because, in this study, only the fronds of Durvillaea incurvata were analyzed. In contrast, previous data refer to the entire seaweed, which includes the part that has a higher amount of insoluble fiber. Table 1 Proximate composition of Durvillaea incurvata fronds and seaweed-based matrices (CE, MS and SS) (n = 3). Chemical composition (g/100g) Freeze-dried seaweed CE MS SS Moisture 12.8 9.5 8.3 80.0 Protein 7.0 6.1 9.4 1.1 Fat 0.4 0.4 5.4 0.1 Ash 15.6 13.2 17.0 11.0 Fiber 52.0 19.7 58.0 7.4 Soluble fiber 21.4 20.7 32.0 2.5 Insoluble fiber 30.6 0.0 26.0 5.1 Available carbohydrates 12.2 50.1 1.9 0.4 Following the proximate analyses of the samples and their raw material, the phlorotannin content was determined. The TSPC of CE, MS, and SS was measured before and after in vitro static digestion under standard and older adult gastrointestinal conditions (Table 2 ). Before digestion, CE exhibited the highest TSPC (22.5 mg PhE/g dw), while SS had the lowest value (6.2 mg PhE/g dw). This variation can be attributed to the processing conditions of seaweed-based products. The same behavior was observed after in vitro digestion, independently of the gastrointestinal conditions (Table 2 ). The TSPC of CE significantly ( p < 0.05) increased (10-fold) after simulated standard digestion, reaching 226.5 mg PhE/g dw. This increase was less pronounced in the older adult model (3-fold), where TSPC reached 74.8 mg PhE/g dw. Similarly, the TSPC of MS (11.6 mg PhE/g dw) increased significantly ( p < 0.05) after simulated digestions, achieving 5-fold and 4-fold increases for standard and older adult conditions, respectively. The bioaccessibility of TSPC in brown seaweed-based products was compared using the two digestion models and expressed as the recovery index (Fig. 3 a). For CE, the recovery index of TSPC was significantly higher ( p < 0.05) in the standard model (1005%) compared to the older adult assay (332%). The recovery index of the MS for the standard model was 468%, slightly higher than the 434% observed for older adult conditions. On the other hand, SS showed the lowest bioaccessibility values, especially under the older adult model conditions (406% for the standard model and 152% for the older adult model). Even though both digestion models significantly increased the TSPC in all three seaweed-based matrices, the older adult model resulted in significantly lower TSPC bioaccessibility values ( p < 0.05). In this study, the analyzed food matrices, obtained through different processing techniques, exhibited varying structures and moisture content at the time of digestion. These differences significantly influenced both the initial concentrations of phlorotannins and their subsequent release during gastrointestinal digestion. For the specific case of CE, it was obtained through USAE at low temperature (40°C), which preserved a higher polyphenol content. In contrast, SS and MS were processed at higher temperatures (~ 100°C), which may have contributed to a reduction in TSPC. Processing techniques such as micronization and USAE appear to play a pivotal role in extracting and enhancing the bioaccessibility of phlorotannins (Ribas-Agustí et al., 2018 ). CE was obtained through a USAE, which is reported to be more effective in extracting phlorotannins as the acoustic cavitation improves solvent penetration into the tissue, enhancing the release of these compounds from the matrix (Ummat et al., 2020 ). MS was developed through micronization, which increases the extractability and bioaccessibility of polyphenols by reducing the particle size of the food material via mechanical and high-shearing processes. However, the covalently bound polyphenols remain difficult to release (Speroni et al., 2021; Speroni et al., 2019 ). Interestingly, TSPC was higher after digestion for the two evaluated in vitro models (Fig. 3 ). However, the increase was lower in the digestion model for older adults (Fig. 3 a), probably due to the higher gastric pH conditions, which partially hinder the release of polyphenols from the food matrix. Recent studies have documented these changes, reporting a decrease in polyphenol bioaccessibility in apples (up to 40%) and chia seeds (up to 59%) during in vitro digestion using elderly condition models (Hernández-Olivas et al., 2023 ; Shang et al., 2022 ). Our results are consistent with previous studies that reported enhanced polyphenols bioaccessibility following gastrointestinal digestion, reaching values above 100% in different fruit juices (up to 520%) and seaweed species (up to 401%) (Attri et al., 2017 ; Subbiah et al., 2024 ). Particularly, Subbiah et al. ( 2024 ) observed a significant enhancement in the bioaccessibility of phenolic compounds in five Australian beach-cast seaweed species, including Durvillaea sp , which exhibited high flavonoid content in the small intestinal phase and high tannin content in the gastric phase. In contrast, Corona et al. ( 2017 ) found that in vitro digestion and fermentation significantly decreased the polyphenolic profile in brown seaweed extract, increasing the low molecular weight forms of phlorotannin-rich extracts from Ascophyllum nodosum . The variability of these results may be related to the synergistic or antagonistic antioxidant effects between marine polysaccharides and phenolic compounds, which are determined by structural compatibility and the relative ratios of polysaccharides to phenolics (Lee et al., 2025 ). In some cases, the gastrointestinal tract acts as a polyphenol extractor by breaking down solid food structures through the mechanical and chemical actions of the oral and digestive phases, respectively (Parada & Aguilera, 2007 ; Tagliazucchi et al., 2012 ). However, depending on the overall composition of the food matrix, it could change. Other studies have observed a significant reduction in polyphenol recovery in the gut phase, likely due to interactions with dietary macronutrients such as dietary fiber, minerals, or proteins, as well as degradation during digestion, which can decrease their solubility, bioaccessibility, and efficacy (Correa-Betanzo et al., 2014 ; McDougall et al., 2005 ). In this study, we hypothesized that the observed increase in TSPC during digestion is likely associated with the release of macromolecular or non-extractable polyphenols previously bound to the dietary fiber fraction of seaweeds, which plays a key role in limiting their initial extractability (Pérez-Jiménez et al., 2013 ). Table 2 Changes in the total contents of soluble polyphenols (TSPC), phlorotannins (PhT), and antioxidant capacity (DPPH and ORAC methods) of seaweed-based matrices (CE, MS and SS) after in vitro gastrointestinal digestion Parameter Brown seaweed-based matrices Undigested Digested (Standard model) Digested (Older adult model) TSPC (mg PhE /g dw) CE 22.5 ± 0.0 aA 226.5 ± 0.4 aC 74.8 ± 0.6 aB MS 11.6 ± 1.2 bA 54.3 ± 0.2 bC 50.4 ± 2.4 bB SS 6.2 ± 0.0 cA 25.3 ± 0.0 cC 9.5 ± 0.4 cB PhT (mg PhE /g dw) CE 0.8 ± 0.0 aA 1.0 ± 0.1 aA 0.9 ± 0.4 aA MS 0.2 ± 0.0 bA < 0.1 ± 0.0 bB < 0.1 ± 0.0 bB SS < 0.1 ± 0.0 c < 0.1 ± 0.0 b < 0.1 ± 0.0 b DPPH (umol T eq/g dw) CE 15.6 ± 0.0 bA 26.8 ± 0.1 aB 15.0 ± 6.8 aA MS 42.7 ± 0.3 aA 2.5 ± 0.8 bB 1.9 ± 0.3 bB SS 10.5 ± 0.0 cA 0.5 ± 0.1 cC 0.7 ± 0.1 bB ORAC (umol T eq/g dw) CE 101.0 ± 7.7 bA 529 ± 64 aB 511 ± 85 aB MS 62.6 ± 1.2 cA 217.6 ± 4.1 bB 482 ± 75 aC SS 128.0 ± 12.0 aA 139.2 ± 4.8 cA 165.8 ± 6.7 bB The results represent the mean of three repetitions with their standard deviation. A,b,c Different lowercase letters indicate significant differences ( p < 0.05) between seaweed-based products (within the column). A, B, C Different capital letters indicate significant differences ( p < 0.05) between digestion models (within a row). The initial PhT of the three seaweed-based matrices exhibited notable differences (Table 2 ). The CE showed the highest baseline concentration (0.8 mg PhE/g dw), followed by the MS with 0.2 mg PhE/g dw, and the SS, with a very low value (< 0.1 mg PhE/g dw). These variations are primarily attributed to the processing conditions, particularly temperature, with CE being the only matrix obtained through low-temperature USAE extraction at 40°C, which helps preserve phlorotannins. After in vitro digestion, CE was the only matrix that maintained PhT bioaccessibility above 100%, with a recovery index of 130% under standard conditions and 112% in the older adult model. In the specific case of CE, the PhT values likely correspond to the phlorotannins previously identified in this extract, including carmalols, fucols, and eckols. The cleavage of their characteristic aryl–ether and aryl–aryl linkages under gastrointestinal conditions may lead to their release in more soluble forms, which could partly account for the higher PhT levels observed after digestion (Rivera-Tovar et al., 2025 ). In contrast, MS and SS exhibited a significant decrease ( p < 0.05 ) in PhT, with much lower bioaccessibility values, ranging from 28% to 42% for MS and 36% to 51% for SS, regardless of the digestion model applied (Table 2 , Fig. 3 b). This suggests that matrix composition plays a key role in phlorotannin release, as MS and SS likely retained phlorotannins within more complex matrices that limited their extractability and digestion-driven release (Tarko & Duda-Chodak, 2020 ). The in vitro gastrointestinal digestion study showed an antagonistic antioxidant effect in MS and SS matrices, attributed to the ability of marine polysaccharides to trap phenolic compounds. Correspondingly, previous research has highlighted that benzene rings and -OH groups from phlorotannins can non-covalently interact through hydrogen bonding and Van der Waals forces with the sulfate and hydroxyl groups of fucoidan, a primary polysaccharide in brown seaweed (Lee et al., 2025 ; Liu & Le Bourvellec, 2023 ). This effect becomes more pronounced in MS and SS due to their higher concentrations of polysaccharides and crude fibers (Table 1 ), which influence the release of phenolic compounds under simulated gastrointestinal conditions. Phlorotannins are firmly bound to cellular and structural polysaccharides, such as fucoidans and alginates, that can form a three-dimensional gel-like network that captures phlorotannins (Liu & Le Bourvellec, 2023 ). Integrating all previous values and considering that the recommended serving sizes for these products differ significantly, Fig. 4 illustrates the differences in TSPC and PhT among the three D. incurvata- based products (CE, MS, and SS) based on recommended serving sizes. Notably, the CE, administered at 1 g of lyophilized extract, exhibited the highest initial TSPC (22 mg PhE) and PhT (0.7 mg PhE). In contrast, MS and SS, despite their larger serving sizes (1.6 g and 80 g, respectively), showed lower initial values for both TSPC and PhT. Following the in vitro digestion, substantial differences were observed in the bioaccessible polyphenols, depending on the food matrix and digestive model. The serving size of CE displayed a remarkable increase in TSPC post-digestion, reaching 227 mg PhE under standard conditions and 75 mg PhE in the older adult model. Conversely, the MS and SS products exhibited lower polyphenol recoveries. However, a notable enhancement was observed for the serving size of SS under the standard model (405 mg PhE) due to its larger initial mass (Fig. 4 ). CE consistently maintained higher values than MS and SS regarding phlorotannins, reflecting its high potential as a functional food ingredient. 3.4. Stability of the antioxidant capacity of seaweed-based matrices An evaluation of antioxidant capacity complemented previous measurements of phlorotannin content as a first step in predicting their biological activities. The DPPH assay results (Table 2 ) revealed significant differences in antioxidant capacity among the undigested seaweed-based samples. MS presented the highest scavenging capacity, with a value of 42.7 ± 0.3 µmol T eq/g dw, followed by CE at 15.6 ± 0.1 µmol T eq/g dw. SS exhibited the lowest DPPH scavenging capacity, with a 10.5 ± 0.1 µmol T eq/g dw. As previously observed for other vegetable-based matrices, DPPH scavenging capacities of CE, MS, and SS were not entirely associated with TSPC or PhT. This is likely because DPPH capacity depends on the molecular structure and reactivity of polyphenols rather than solely on their concentration (Tierney et al., 2013 ). Processing methods, such as micronization or low-temperature USAE, can also influence antioxidant capacity by altering the accessibility and effectiveness of polyphenols in neutralizing free radicals. Thus, even with varying polyphenol concentrations, molecular structure and processing conditions contribute to differences in DPPH scavenging capacity (Rodríguez-Roque et al., 2016 ). Standard digestion conditions significantly increased ( p < 0.05) the DPPH values of the undigested CE sample, increasing it from 15.6 µmol T eq/g to 26.8 µmol T eq/g dw (2-fold). However, digestion under older adult conditions did not cause an increase in this indicator (15.0 µmol T eq/g dw). Among the three seaweed-based matrices, undigested MS showed the highest DPPH scavenging capacity (42.7 µmol T eq/g dw), which decreased substantially following digestion under standard (2.5 µmol T eq/g dw) and older adult (1.9 µmol T eq/g dw) conditions. Similar to MS, DPPH values of SS significantly decreased 22-fold and 14-fold after digestion under standard (from 10.5 µmol T eq/g dw to 0.5 µmol T eq/g dw) and older adults (from 10.5 µmol T eq/g dw to 0.7 µmol T eq/g dw) simulated conditions, respectively. Nevertheless, the older adult digestion model had significantly less impact on the decrease in the SS DPPH scavenging capacity. The modifications in DPPH values were also expressed in terms of the recovery index to evaluate the stability of this in vitro bioactivity descriptor (Fig. 5 a). The DPPH recovery index of CE increased by up to 171% after in vitro digestion under standard conditions, while 96% of its antioxidant capacity was retained in older adult samples. MS and SS presented a significant loss of their antioxidant capacity after digestion, maintaining 4% and 7% of the DPPH values, respectively, regardless of the digestion model applied (Fig. 5 a). The DPPH assay evaluates antioxidant capacity based on the ability of compounds to donate hydrogen atoms, a function strongly dependent on the presence and accessibility of hydroxyl (-OH) groups. In this study, CE presented the highest DPPH scavenging capacity after digestion, probably due to the presence of high-molecular-weight phlorotannin oligomers that, upon digestion, released smaller molecules with accessible hydroxyl groups. These smaller, more reactive molecules exhibited a greater interaction with the DPPH radical, resulting in a higher apparent antioxidant capacity (Prior et al., 2005 ). This finding aligns with our previous study (Rivera-Tovar et al., 2025 ), which identified the presence of high-molecular-weight phlorotannin oligomers in the D. incurvata extract. In contrast, the MS and SS samples contained higher amounts of low-solubility carbohydrate oligomers, which likely formed interactions with phlorotannins, making them resistant to digestion (Parada et al., 2019 ). This binding effect reduced the accessibility of hydroxyl groups, thereby hindering their ability to neutralize DPPH radicals. Consequently, even though these matrices may contain phenolic compounds, their radical-scavenging capacity was diminished due to steric hindrance and reduced availability of functional groups essential for the reaction. A similar trend to that observed for TSPC was found when comparing the DPPH values after the standard and older adult digestion models. This reduction is likely due to higher gastric pH and longer digestion times, which limit the release of polyphenols. These results highlight the impact of age-related digestive changes on the availability of antioxidants. Considering the phlorotannins tentatively identified in the CE in a previous study (Rivera-Tovar et al., 2025 ), the antioxidant capacity was also assessed using the ORAC assay (Fig. 5 b), which has been suggested to be more selective when the extract contains high-molecular-weight polyphenols (Rivera-Tovar et al., 2021 ). SS showed the highest antioxidant capacity, with a value of 128 ± 12 µmol T eq/g dw, followed by CE with a moderate ORAC value of 101.0 ± 7.7 µmol T eq/g dw, while MS presented the lowest ORAC value at 62.6 ± 1.2 µmol T eq/g dw (Table 2 ). The ORAC values for digested samples (Fig. 5 b) showed that digestion markedly enhanced ( p < 0.05) the antioxidant capacity of CE, MS, and SS, with values of 101.0 µmol T eq/g dw, 62.6 µmol T eq/g dw, and 127.7 µmol T eq/g dw, respectively. This suggests that antioxidant compounds remain stable and effective after digestion, regardless of the digestive model employed. Under standard digestion conditions, the ORAC value of CE significantly increased ( p < 0.05) to 529.1 µmol T eq/g dw (5-fold), MS ORAC to 217.6 µmol T eq/g dw (3-fold), and SS ORAC to 139.2 µmol T eq/g dw (1.1-fold) (Fig. 5 b). The digestive conditions model for older adults slightly decreased the ORAC value of CE, with no significant differences compared to the standard digestion model. Nevertheless, the bioaccessibility index of the antioxidant ORAC capacity of MS and SS was significantly enhanced ( p < 0.05), particularly in MS (up to 482.3 µmol T eq/g dw and 165.8 µmol T eq/g dw, respectively), which showed a value 8-fold higher than its undigested counterpart. According to the ORAC results, the CE and MS were the matrices with the highest potential bioactivity, showing a substantial increase in their bioaccessibility indexes (Fig. 4 b). Under standard digestion conditions, the CE, MS, and SS showed increases in their bioaccessibility indexes of 524%, 347%, and 109%, respectively. Furthermore, the older adult digestion model appears to have a distinct impact on the ORAC bioaccessibility index, particularly for MS, where the bioaccessibility index is significantly higher (770%) than in the standard model. In contrast, SS remained the least effective in terms of the ORAC bioaccessibility index, although it improved under the older adult digestion model (130%). These results underscore the complex, non-linear relationship between the phlorotannin concentration in the three seaweed-based carriers and their antioxidant capacity. CONCLUSIONS This study highlights that the obtaining process, matrix composition, and age-related digestive changes are key determinants of the bioaccessibility of phlorotannins in brown seaweed-based products. The CE, which can be considered a safe and sustainable food ingredient with antioxidant capacity obtained through low-temperature USAE, showed higher phlorotannin stability and bioaccessibility compared to commercially available products obtained through higher-temperature processes (MS and SS). Furthermore, simulated digestive conditions for older adults revealed significant variations in phlorotannin bioaccessibility, likely due to age-related physiological changes such as reduced enzyme activity and altered gastric pH. The antioxidant capacity of seaweed-based products increased significantly following digestion; however, it remained unaffected by alterations related to the older adult model, thereby confirming that seaweed is a promising raw material for developing functional foods targeted at the elderly. Future studies should perform a more detailed analysis of the chemical and structural features of antioxidant compounds in brown seaweed, considering that other compounds with antioxidant capacity, such as sulfated carbohydrates, might also be present in USAE extracts. Furthermore, clinical studies are necessary to verify the bioavailability of brown seaweed phlorotannins in older adults, including the potential contribution of the colonic microbiota, as well as to evaluate the long-term health effects of seaweed-based products, particularly for aging populations. Declarations ETHICS DECLARATION Not applicable. FUNDINGS ANID-Chile funded this research through the FONDECYT Regular grants 1220097 and 1230115, FONDECYT Iniciación grant 11230112, and the FOVI grant 240263. Author Contribution MAR: Conceptualization, data curation, figure preparation, and manuscript writing. GRA, LS: Experimental work and data analysis; LS also participated in manuscript editing. 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Elsevier Ltd. https://doi.org/10.1016/j.tifs.2023.104301 Additional Declarations No competing interests reported. Supplementary Files SupplementarymaterialS2.docx Cite Share Download PDF Status: Published Journal Publication published 28 Feb, 2026 Read the published version in Food Production, Processing and Nutrition → 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":508315,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of \u003cem\u003ein vitro\u003c/em\u003e digestion models (standard and older adult) for assessing phlorotannin bioaccessibility and antioxidant capacity stability of \u003cem\u003eDurvillaea incurvata\u003c/em\u003e-based products. SSF: simulated saliva fluid; SGF: simulated gastric fluid; SIF: simulated intestinal fluid.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7427244/v1/279264ca9c731eb81b75f4e5.png"},{"id":92732552,"identity":"4ceb355b-ce80-4c34-ad5b-1ad862fb6740","added_by":"auto","created_at":"2025-10-03 16:08:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":290756,"visible":true,"origin":"","legend":"\u003cp\u003eMTS assay on the effect of \u003cem\u003eD. incurvata\u003c/em\u003e CE at different concentrations on human kidney non-tumoral HEK293T (a) and human colon cancer HT29 cell lines (b). Different letters represent statistically significant differences between concentrations (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7427244/v1/8ce5e0606b0b691a7b819335.png"},{"id":92732104,"identity":"d14c8162-2126-4a24-a806-76b02df6d6ab","added_by":"auto","created_at":"2025-10-03 16:00:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":470645,"visible":true,"origin":"","legend":"\u003cp\u003eBioaccessibility of TSPC (a) and PhT (b) of seaweed-based matrices (CE, MS and SS) using standard and older adult \u003cem\u003ein vitro\u003c/em\u003e digestion models. \u003csup\u003eA,b,c\u003c/sup\u003e Different lowercase letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) between vehicles. \u003csup\u003eA, B, C\u003c/sup\u003e Different capital letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) between digestion models.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7427244/v1/83ad1cea69c8c711d1ca362e.png"},{"id":92732109,"identity":"45f93679-063f-4462-8e39-0219d20fe0e4","added_by":"auto","created_at":"2025-10-03 16:00:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":429705,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of food processing conditions, food composition and digestion model on the quantity of phlorotannins accessible from a recommended serving of seaweed-based products post-digestion.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7427244/v1/7d28a878fa5c150e876b5c52.png"},{"id":92732556,"identity":"866872f7-d7df-41d0-8af0-057f84cd8809","added_by":"auto","created_at":"2025-10-03 16:08:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":475450,"visible":true,"origin":"","legend":"\u003cp\u003eStability of antioxidant capacity measured by DPPH (a) and ORAC (b) assays in three seaweed-based products (seaweed crude extract: CE, micronized seaweed: MS, and seaweed salad: SS) after \u003cem\u003ein vitro\u003c/em\u003e digestion using standard and older adult digestion models.\u003csup\u003e a,b,c\u003c/sup\u003e Different lowercase letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) between vehicles. \u003csup\u003eA,B,C\u003c/sup\u003e Different capital letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) between digestion models.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7427244/v1/65520abb1efbb729ecffd4a5.png"},{"id":103765652,"identity":"124fbf78-6644-49bf-bbed-d48f711121b5","added_by":"auto","created_at":"2026-03-02 16:06:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2921799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7427244/v1/4c56f29a-5581-49f5-8b8b-7ef1690b5ea0.pdf"},{"id":92732106,"identity":"a026374e-d86a-4ea1-85bb-7ecc01d2c65b","added_by":"auto","created_at":"2025-10-03 16:00:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16974,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7427244/v1/86cb75838cfd8550a5ccaae0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of phlorotannin bioaccessibility and antioxidant capacity stability of a safe and sustainable brown seaweed extract: role of age-related digestion changes and matrix composition","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe global population is aging rapidly, with estimates suggesting that by 2050, individuals aged 60 and above will constitute nearly 22% of the worldwide population. This demographic shift brings with it significant health challenges, particularly for older adults, who are prone to various chronic diseases such as cardiovascular diseases, diabetes, and osteoporosis (WHO, 2024). Among the less visible but equally impactful health concerns for older populations are gastrointestinal issues, which can lead to malabsorption and nutritional deficiencies (Arazo-Rusindo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bustos-Arriagada et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Digestive problems, including reduced stomach acid production, slower gut motility, and altered gut microbiota composition, are common in older individuals. They can significantly impair the absorption of essential nutrients, leading to deficiencies in vitamins, minerals, and proteins (Dumic et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Menard et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These deficiencies can exacerbate age-related frailties, increasing the risk of muscle wasting (sarcopenia), bone demineralization, and immune system decline (Gallo et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGiven these health concerns, it has become increasingly important to design tailored food products that specifically meet the nutritional needs of older adults. Customized nutrition for the elderly involves addressing not only the macronutrient and micronutrient requirements but also integrating bioactive compounds, such as polyphenols, into food matrices that can help mitigate the onset of chronic diseases. Incorporating polyphenols into functional foods for older adults presents a viable approach to address the digestive and chronic disease-related challenges of this demographic, particularly given the increasing evidence on the role of these compounds in improving intestinal health through various mechanisms of action (Esp\u0026iacute;n et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, ensuring the post-digestion bioavailability of these compounds is crucial.\u003c/p\u003e\u003cp\u003ePhlorotannins, a specific class of polyphenols found in brown seaweed, have emerged as up-and-coming candidates for functional food and pharmaceutical applications due to their antioxidant (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Erpel et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rajauria et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), antidiabetic (Gheda et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), anti-inflammatory (Besednova et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; S. Lee et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and anti-carcinogenic properties (Catarino et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cotas et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), although clinical trials with these compounds are still scarce (Keleszade et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In parallel, brown seaweeds are recognized as low-impact food ingredients due to their rapid growth, minimal freshwater and fertilizer requirements, and their capacity to absorb carbon dioxide during cultivation. These characteristics establish seaweeds as promising sustainable resources within the framework of climate change and resource-limited food systems (Cherry et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this sense, different studies have evaluated sustainable extraction processes, such as ultrasound-assisted extraction, to obtain natural extracts from seaweed, which contain phloroglucinol polymers and mannitol (Mu\u0026ntilde;oz-Molina et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Rivera-Tovar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Nevertheless, the use of phlorotannin-rich seaweed products remains limited (Pacheco et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), partly due to concerns regarding seaweeds' potential to accumulate toxic heavy metals (Erpel et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, it is essential to ensure that seaweed-based ingredients contain safe levels of these harmful compounds before including them in food products at non-cytotoxic concentrations.\u003c/p\u003e\u003cp\u003eBioaccessibility and bioavailability are key aspects to consider when using polyphenols as dietary ingredients, given the extent of their metabolic fate after intake, ultimately resulting in final bioactive metabolites (Cort\u0026eacute;s-Mart\u0026iacute;n et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, little information exists on how phlorotannins transform after digestion and their resulting biological activity (Corona et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Subbiah et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, recent studies have shown that the age-related changes in the digestion process can decrease the bioavailability of phenolic compounds, as observed after \u003cem\u003ein vitro\u003c/em\u003e digestion of apples or chia seeds (Hern\u0026aacute;ndez-Olivas et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, the food matrix plays a crucial role in regulating the release and absorption of these bioactive compounds during the digestion process (Qazi et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Y. Zhang et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Meeting the nutritional needs of the aging population requires a comprehensive approach that combines delivering essential nutrients with including bioactive compounds, such as phlorotannins, while also evaluating their bioaccessibility and identifying factors that affect their absorption in the intestine.\u003c/p\u003e\u003cp\u003eThis study investigates the bioaccessibility of phlorotannins and the stability of their antioxidant capacity after simulated gastrointestinal digestion, considering the physiological changes associated with aging. It evaluates three \u003cem\u003eDurvillaea incurvata\u003c/em\u003e-based matrices produced through various processing methods: a fresh seaweed salad (SS), micronized seaweed (MS), and an innovative seaweed extract (CE) containing phlorotannins (trimers to octamers of phloroglucinol) and mannitol, specifically designed to address issues of sustainability, efficacy, and safety.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003eTwo commercially available \u003cem\u003eDurvillaea incurvata\u003c/em\u003e foods (MS and SS) were compared with a CE obtained through a sustainable and scalable process to evaluate their potential as phlorotannin oral delivery systems. The CE had been previously found to contain mannitol and phlorotannins with structural features associated with anti-inflammatory activity (Rivera-Tovar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Safety was first assessed by determining the heavy metal content of both raw material and CE, followed by a cytotoxicity assay to establish a safe dose for use. The proximate composition of CE, MS, and SS was also analyzed. After completing safety assessment and preliminary chemical characterization, the three products were subjected to in vitro digestion using two INFOGEST 2.0 protocols: the standard model (Brodkorb et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and an adaptation representing older adults (Menard et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), to evaluate phlorotannin bioaccessibility and antioxidant stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2.1.1. \u003cem\u003eDurvillaea Incurvata\u003c/em\u003e samples\u003c/h2\u003e\u003cp\u003e\u003cem\u003eDurvillaea incurvata\u003c/em\u003e fronds used for the extract were collected from the coast of Concepci\u0026oacute;n, Chile (36\u0026deg;32\u0026prime;55\u0026Prime;S 72\u0026deg;56\u0026prime;10\u0026Prime;W) during the fall of 2023. Seaweed Place SPA, Chile, kindly provided the MS and SS.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.1.2. Chemicals and analytic reagents\u003c/h2\u003e\u003cp\u003eDistilled water, methanol (99.9%, HPLC grade), acetone (99.8%, HPLC grade), and acetic acid (99.0%), sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e), dipotassium phosphate (K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), potassium dihydrogen phosphate (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), ammonium carbonate ((NH\u003csub\u003e4\u003c/sub\u003e)2CO\u003csub\u003e3\u003c/sub\u003e), calcium chloride dihydrate (CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO), magnesium chloride hexahydrate (MgCl\u003csub\u003e2\u003c/sub\u003e \u0026middot; 6H\u003csub\u003e2\u003c/sub\u003eO), potassium chloride (KCl), potassium dihydrogen phosphate (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), sodium chloride (NaCl) and sodium hydroxide (NaOH), hydrochloric acid 37% (HCl), α-amylase from porcine pancreas (1.25\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e kat/mL), pepsin from porcine gastric mucosa (3.33 \u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e kat/mL), pancreatin from porcine pancreas (as trypsin activity 1.67\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e kat/mL) and porcine bile salts (7.07 g/100 mL), were purchased from Merck LifeScience S.L.U. (Santiago, Chile). The Folin\u0026ndash;Ciocalteu reagent, DPPH (1,1-Diphenyl-2-picryl-hydrazil), fluorescein sodium salt, AAPH (2,2\u0026acute;-azobis-2-methylpropionamidine dihydrochloride), standards of phloroglucinol (99%) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Sigma-Aldrich (Steinem, Germany).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Methods\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Obtaining a sustainable crude extract (CE) from \u003cem\u003eDurvillaea incurvata\u003c/em\u003e.\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eDurvillaea incurvata\u003c/em\u003e CE was obtained using ultrasound-assisted extraction (USAE). The fronds were chopped, freeze-dried, ground, and sieved to obtain particles smaller than 710 \u0026micro;m. A 20 g sample was mixed with 200 mL of 32.5% ethanol/water solution (Rivera-Tovar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). USAE was performed with an ultrasonic probe (BIOBASE, China) at 20 kHz, 40% power (600 W), 30\u0026deg;C, and a pulse cycle of 5/15 seconds. The mixture was centrifuged at 2490\u0026times;g for 5 minutes, and the liquid phase was concentrated by rotary evaporation at 40\u0026deg;C for 90 minutes. The resulting extract was freeze-dried at -80\u0026deg;C for 24 hours to produce a stable powder. The CE used in this study was previously characterized in detail by our group (Rivera-Tovar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In that work, FTIR analysis confirmed the presence of functional groups associated with both polyphenols and mannitol, while HPLC-IR quantification indicated a mannitol content of 259\u0026thinsp;\u0026plusmn;\u0026thinsp;24 mg/g dried extract. Additionally, UHPLC-QToF-MS enabled the tentative identification of phlorotannins ranging from trimers to octamers, including carmalols, fucols, and eckols.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Heavy metals analysis\u003c/h2\u003e\u003cp\u003eTo evaluate the safety of both the dried seaweed and its CE, concentrations of arsenic (As), cadmium (Cd), lead (Pb), mercury (Hg), and tin (Sn) were quantified following the methodology outlined by (Erpel et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The quantification employed two complementary analytical techniques: atomic absorption spectroscopy (AAS) for As and Hg and inductively coupled plasma atomic emission spectroscopy (ICP-AES) for Cd, Pb, and Sn. Analyses of the dried seaweed samples adhered to the standardized procedures TMECC 04.14 and 04.12-B (Thompson et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). For the CE samples, evaluations followed the ME-12 protocol (Superintendencia de Servicios Sanitarios, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), supplemented by Standard Methods 3030C and E, and 3120B (APHA et al., 1999). All results were expressed in milligrams of metal per kilogram of dry weight (mg/kg DW), with each analysis conducted in duplicate to ensure analytical accuracy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. Cytotoxicity assay\u003c/h2\u003e\u003cp\u003eThe effect of CE on cell viability was determined using the MTS assay as described previously (Sim\u0026oacute;n et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Briefly, 3 \u0026times; 10^3 HEK293T and HT29 cells were seeded in 96-well plates. After an initial 24-hour period in culture, cells were treated with increasing concentrations of CE (0\u0026ndash;4 g/100 mL) for 48 hours. Viability was measured according to the manufacturer\u0026rsquo;s instructions (Cat. #G3580, Promega, Madison, WI).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4. Proximate analysis\u003c/h2\u003e\u003cp\u003eThe proximate composition of \u003cem\u003eDurvillaea incurvata\u003c/em\u003e and its three based products (CE, MS, and SS) was assessed. Moisture content was measured using the thermogravimetric method at 105\u0026deg;C in an oven, by the Chilean standard NCh 841.Of78 (INN, 1978). Protein content was determined using the Kjeldahl method, as specified in the Chilean standard NCh 2748. Of.2019 (INN, 2019), with a nitrogen-to-protein conversion factor of 6.25. Oil content was measured through ether extraction in a Soxhlet apparatus according to AOAC Official Methods of Analysis, method 920.39 (AOAC International, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Ash content was obtained by incineration at 550\u0026deg;C in a muffle furnace, following AOAC Official Method 942.05 (AOAC International, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Total, soluble, and insoluble dietary fiber were analyzed using the enzymatic-gravimetric method, as outlined in AOAC Official Method 991.43 (AOAC International., 2016). Available carbohydrates were calculated by difference based on AOAC Official Method 986.25 (AOAC International., 2012).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5. Determination of phlorotannin content\u003c/h2\u003e\u003cp\u003eThe phlorotannin content of both undigested and digested samples was determined by evaluating their total soluble polyphenol content (TSPC) and total phlorotannin content (PhT).\u003c/p\u003e\u003cp\u003e\u003cem\u003eTSPC\u003c/em\u003e was measured using the method of (Singleton \u0026amp; Rossi, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). Briefly, 20 \u0026micro;L of the standard extracts or extraction solution (blank) were sequentially mixed with 10 \u0026micro;L of Folin-Ciocalteu reagent (diluted 1:2 with deionized water) and 150 \u0026micro;L of deionized water. The solution was homogenized, and 20 \u0026micro;L of a 10% Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e solution (w/v) was added. The mixture was left to react in the dark at room temperature for one hour. Absorbance readings were recorded at 750 nm using a multi-mode microplate reader (Biotek Instruments, Winooski, VT, USA) and compared to a phloroglucinol calibration curve (15\u0026ndash;150 mg/L). Each analysis was performed in triplicate, and the results were expressed as mg of phloroglucinol equivalents (mg PhE) per g of dry sample.\u003c/p\u003e\u003cp\u003ePhT was determined using the DMBA method according to the protocol developed by (Rivera-Tovar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Samples were reconstituted (20 mg in 10 mL of water) and analyzed in duplicate. The reaction involved shaking for 30 minutes, followed by incubation at 25\u0026deg;C in the dark for 60 minutes. Absorbance was measured at 515 nm using a 96-well microplate reader (Biotek Instruments, Winooski, VT, USA). Values were interpolated from a phloroglucinol calibration curve (1\u0026ndash;40 mg/L) and expressed as milligrams of phloroglucinol equivalents (mg PhE) per g of dry sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6. Determination of the antioxidant capacity\u003c/h2\u003e\u003cp\u003eAntioxidant capacity was assessed using two complementary assays: a modified version of the 1,1-Diphenyl-2-picryl-hydrazil scavenging capacity (DPPH) method and an adjusted version of the Oxygen Radical Antioxidant Capacity (ORAC) method. DPPH assay was developed as follows: methanolic extracts or Trolox standards (100 \u0026micro;L) were mixed with 100 \u0026micro;L of DPPH solution (0.048 mg/mL) in 96-well plates. After 30 min of incubation in the dark at room temperature, absorbance was measured at 520 nm (Biotek, USA) (Brand-Williams et al., 1995). Blanks were included to correct for sample color. Results were calculated from a Trolox standard curve (1\u0026ndash;32 \u0026micro;M) and expressed as \u0026micro;mol Trolox equivalents per gram of dry sample. Analyses were performed in triplicate. The ORAC microassay was performed as described by (Ou et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), with slight adjustments. In a 96-well microplate, 150 \u0026micro;L of a solution containing 96 nM of fluorescein in a 75 mM phosphate buffer (pH 7.4) was combined with 25 \u0026micro;L of either extract or a diluted Trolox standard. The plate was incubated for 30 min at 37\u0026deg;C. The reaction was initiated by adding 50 \u0026micro;L of a 79 mM AAPH solution prepared in the phosphate buffer. Fluorescence readings were taken every minute for 2 hours at 37\u0026deg;C using a multi-mode microplate reader (Biotek Instruments, Winooski, VT, USA), with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The area under the curve (AUC) for samples, standards, and phosphate buffer blanks (AUC\u003csub\u003eblank\u003c/sub\u003e) was determined using Gen5 Data Analysis Software (Biotek Instruments). The net AUC values for both samples and standards were calculated based on [Eq.\u0026nbsp;2]:\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{N}\\text{e}\\text{t}\\:\\text{A}\\text{U}\\text{C}={\\left[AUC\\right]}_{sample\\:or\\:Std}-{\\left[AUC\\right]}_{blank}\\)\u003c/span\u003e\u003c/span\u003e [Eq.\u0026nbsp;2]\u003c/p\u003e\u003cp\u003eThe ORAC values of the samples were calculated by interpolating from the standard curve of Net AUC\u003csub\u003eStd\u003c/sub\u003e vs. Trolox concentrations (1\u0026ndash;32 \u0026micro;M). Results were reported as \u0026micro;mol of Trolox equivalents (\u0026micro;mol T eq) per g of dry sample. All analyses were conducted in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.2.7. \u003cem\u003eIn vitro\u003c/em\u003e gastrointestinal digestion models\u003c/h2\u003e\u003cp\u003eCE, MS, and SS samples were digested using an \u003cem\u003ein vitro\u003c/em\u003e gastrointestinal system based on the INFOGEST 2.0 protocol under standard conditions (Brodkorb et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and an adapted version that simulated the physiological conditions of older adults (Menard et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The digestion process consisted of three phases: oral, gastric, and intestinal, utilizing simulated salivary (SSF), gastric (SGF), and intestinal (SIF) fluids. Fresh SSF, SGF, and SIF ionic electrolyte stock solutions were prepared before the experiments (Supplementary Material S2). After the intestinal phase, the samples were centrifuged at 2490 \u003cem\u003eg\u003c/em\u003e for 5 minutes at 4\u0026deg;C to inactivate the enzymes. A control experiment was also conducted without the food matrix to assess the effect of the chemical environment on the response. The \u003cem\u003ein vitro\u003c/em\u003e bioaccessibility of phlorotannin and antioxidant stability of the liquid fraction (supernatant) of each sample were estimated based on the recovery index (%) (Eq.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{R}\\text{e}\\text{c}\\text{o}\\text{v}\\text{e}\\text{r}\\text{y}\\:\\text{I}\\text{n}\\text{d}\\text{e}\\text{x}\\:\\left(\\text{%}\\right)\\frac{{PC}_{digested\\:sample}\\:or\\:{AA}_{digested\\:sample}}{{PC}_{undigested\\:sample}\\:or\\:{AA}_{undigested\\:sample}}\\text{x}100\\)\u003c/span\u003e\u003c/span\u003e [Eq.\u0026nbsp;1]\u003c/p\u003e\u003cp\u003eRegardless of the digestion model used, the PC\u003csub\u003edigested sample\u003c/sub\u003e corresponds to the TSPC or PhT, and the AA\u003csub\u003edigested sample\u003c/sub\u003e corresponds to the antioxidant capacity values of the three seaweed-based matrices after digestion, respectively. Meanwhile, PC\u003csub\u003eundigested\u003c/sub\u003e and AA\u003csub\u003eundigested\u003c/sub\u003e samples refer to the TSPC, PhT, and the samples' antioxidant capacity values before \u003cem\u003ein vitro\u003c/em\u003e gastrointestinal digestion, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.2.8. Statistical analysis\u003c/h2\u003e\u003cp\u003eResults for viability, TSPC, PhT, radical scavenging capacity (DPPH), and oxygen radical antioxidant capacity (ORAC) were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three replicates per sample. Statgraphics Centurion XVIII Software version 18.1.12 (Stat-Point Technologies Inc., Warrenton, VA, USA) was used for the Analysis of Variance (ANOVA) and Fisher\u0026rsquo;s least significant difference (LSD) test for multiple sample comparisons, with a 95% confidence level (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Shapiro\u0026ndash;Wilk and Levene tests were used to evaluate normality and homoscedasticity, respectively.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Heavy metal content\u003c/h2\u003e\u003cp\u003eDue to concerns about toxic metal concentrations in seaweeds (Prashant et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), which may counteract their benefits, levels in crude \u003cem\u003eD. incurvata\u003c/em\u003e and its CE were measured and compared with authorized values from Chinese and French regulations. It is worth noting that, to date, significant differences and a lack of harmonized international standards or guidelines specifically addressing the food safety of seaweed production persist (Guo et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In China, maximum levels of inorganic arsenic and lead in seaweed and its food products were established by the National Food Safety Standard on Maximum Levels of Contaminants in Foodstuffs (GB 2762\u0026thinsp;\u0026minus;\u0026thinsp;2022) (NHCPRC \u0026amp; SAMR), 2023). For its part, the European Union (EU) has developed specific standards for seaweed-based foodstuffs, setting maximum permissible levels for contaminants such as I, As, Pb, and Cd in food supplements containing between 80% and 100% of dried seaweed (EU, 2018). On the other hand, France was the first European country to establish complete regulation for the maximum levels of heavy metals (inorganic As, Cd, Pb, Hg, and Sn) in seaweed and derived foods (ANSES, 2020).\u003c/p\u003e\u003cp\u003eIn this sense, our results demonstrate the superior safety of CE over raw seaweed (\u003cem\u003eD. incurvata\u003c/em\u003e), which aligns with international efforts to regulate seaweed-derived food products. In particular, Cd content in the raw seaweed (7.6 mg/kg DW) exceeded European and French limits (3.0 mg/kg and 0.5 mg/kg, respectively), whereas it was reduced to below 0.2 mg/kg DW in CE. Similarly, Pb levels decreased from 1.1 to 0.9 mg/kg DW, remaining within acceptable limits for seaweed products. Hg levels in both matrices were well below regulatory limits. Overall, CE revealed a significant reduction of more than 80% in the concentration of heavy metals after the USAE process, with values below the maximum limits recommended by French law. These results align with previous research, which has successfully controlled the toxicity potential of \u003cem\u003eD. incurvata\u003c/em\u003e by applying selective separation techniques, achieving reductions of at least 70% (Erpel et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, it is important to emphasize that reducing heavy metals is crucial not only for toxicological reasons but also to ensure the safe absorption of bioactive compounds, as the presence of these metals can interfere with the bioavailability of polyphenols (Omoarelojie \u0026amp; van Staden, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Toth \u0026amp; Pavia, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Recent studies have confirmed that polyphenols, such as phlorotannins, can bind to heavy metals, forming less bioavailable complexes (Connan \u0026amp; Stengel, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; R. Zhang et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, efficient removal of these metals in the CE is expected to allow these compounds to maintain their functionality during \u003cem\u003ein vitro\u003c/em\u003e digestion.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Sub-cytotoxic concentration\u003c/h2\u003e\u003cp\u003eDue to the uncharacterized cytotoxic potential of this novel CE, it was necessary to verify that it does not impact cell viability. An MTS assay was performed to determine the effect of CE on the human kidney non-tumoral HEK293T and human colon cancer HT29 cell lines. The concentration used for the digestion assay (1 g CE / 100 mL) was sub-cytotoxic for both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), thereby demonstrating the safety of the CE. A higher concentration (4 g CE/100 mL) was only toxic to the colon cancer cell line, highlighting the potential antitumoral effect of this CE.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.3 Changes in phlorotannin levels of seaweed matrices during digestion in both normal and older adult conditions.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOnce the safety of the CE was established, in terms of heavy metal levels and effects on cell viability, the proximate composition of samples was determined (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe macronutrient composition of seaweed fronds was similar to that previously reported by other authors, confirming the nutritional value of this sustainable resource (Burgos-D\u0026iacute;az et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eDurvillaea incurvata\u003c/em\u003e is an excellent source of total fibe\u003cem\u003er (\u003c/em\u003e~\u0026thinsp;50.0%), making it a promising food for preventing several non-communicable diseases (Parada et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Interestingly, the proportion of available carbohydrates and soluble fiber found in this research was higher (5-fold and 2-fold, respectively) than the values previously reported for the same specie (Burgos-D\u0026iacute;az et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This can be explained because, in this study, only the fronds \u003cem\u003eof Durvillaea incurvata\u003c/em\u003e were analyzed. In contrast, previous data refer to the entire seaweed, which includes the part that has a higher amount of insoluble fiber.\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\u003eProximate composition of \u003cem\u003eDurvillaea incurvata\u003c/em\u003e fronds and seaweed-based matrices (CE, MS and SS) (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChemical composition\u003c/p\u003e\u003cp\u003e(g/100g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFreeze-dried seaweed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSS\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMoisture\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e80.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProtein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFat\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAsh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e17.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e52.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e58.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoluble fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e21.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e32.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInsoluble fiber\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e30.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e26.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAvailable carbohydrates\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e12.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.4\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\u003eFollowing the proximate analyses of the samples and their raw material, the phlorotannin content was determined. The TSPC of CE, MS, and SS was measured before and after \u003cem\u003ein vitro\u003c/em\u003e static digestion under standard and older adult gastrointestinal conditions (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Before digestion, CE exhibited the highest TSPC (22.5 mg PhE/g dw), while SS had the lowest value (6.2 mg PhE/g dw). This variation can be attributed to the processing conditions of seaweed-based products.\u003c/p\u003e\u003cp\u003eThe same behavior was observed after \u003cem\u003ein vitro\u003c/em\u003e digestion, independently of the gastrointestinal conditions (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The TSPC of CE significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased (10-fold) after simulated standard digestion, reaching 226.5 mg PhE/g dw. This increase was less pronounced in the older adult model (3-fold), where TSPC reached 74.8 mg PhE/g dw. Similarly, the TSPC of MS (11.6 mg PhE/g dw) increased significantly (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after simulated digestions, achieving 5-fold and 4-fold increases for standard and older adult conditions, respectively.\u003c/p\u003e\u003cp\u003eThe bioaccessibility of TSPC in brown seaweed-based products was compared using the two digestion models and expressed as the recovery index (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). For CE, the recovery index of TSPC was significantly higher (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the standard model (1005%) compared to the older adult assay (332%). The recovery index of the MS for the standard model was 468%, slightly higher than the 434% observed for older adult conditions. On the other hand, SS showed the lowest bioaccessibility values, especially under the older adult model conditions (406% for the standard model and 152% for the older adult model). Even though both digestion models significantly increased the TSPC in all three seaweed-based matrices, the older adult model resulted in significantly lower TSPC bioaccessibility values (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eIn this study, the analyzed food matrices, obtained through different processing techniques, exhibited varying structures and moisture content at the time of digestion. These differences significantly influenced both the initial concentrations of phlorotannins and their subsequent release during gastrointestinal digestion. For the specific case of CE, it was obtained through USAE at low temperature (40\u0026deg;C), which preserved a higher polyphenol content. In contrast, SS and MS were processed at higher temperatures (~\u0026thinsp;100\u0026deg;C), which may have contributed to a reduction in TSPC. Processing techniques such as micronization and USAE appear to play a pivotal role in extracting and enhancing the bioaccessibility of phlorotannins (Ribas-Agust\u0026iacute; et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). CE was obtained through a USAE, which is reported to be more effective in extracting phlorotannins as the acoustic cavitation improves solvent penetration into the tissue, enhancing the release of these compounds from the matrix (Ummat et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). MS was developed through micronization, which increases the extractability and bioaccessibility of polyphenols by reducing the particle size of the food material via mechanical and high-shearing processes. However, the covalently bound polyphenols remain difficult to release (Speroni et al., 2021; Speroni et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInterestingly, TSPC was higher after digestion for the two evaluated \u003cem\u003ein vitro\u003c/em\u003e models (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, the increase was lower in the digestion model for older adults (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), probably due to the higher gastric pH conditions, which partially hinder the release of polyphenols from the food matrix. Recent studies have documented these changes, reporting a decrease in polyphenol bioaccessibility in apples (up to 40%) and chia seeds (up to 59%) during \u003cem\u003ein vitro\u003c/em\u003e digestion using elderly condition models (Hern\u0026aacute;ndez-Olivas et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur results are consistent with previous studies that reported enhanced polyphenols bioaccessibility following gastrointestinal digestion, reaching values above 100% in different fruit juices (up to 520%) and seaweed species (up to 401%) (Attri et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Subbiah et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Particularly, Subbiah et al. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) observed a significant enhancement in the bioaccessibility of phenolic compounds in five Australian beach-cast seaweed species, including \u003cem\u003eDurvillaea sp\u003c/em\u003e, which exhibited high flavonoid content in the small intestinal phase and high tannin content in the gastric phase. In contrast, Corona et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) found that \u003cem\u003ein vitro\u003c/em\u003e digestion and fermentation significantly decreased the polyphenolic profile in brown seaweed extract, increasing the low molecular weight forms of phlorotannin-rich extracts from \u003cem\u003eAscophyllum nodosum\u003c/em\u003e. The variability of these results may be related to the synergistic or antagonistic antioxidant effects between marine polysaccharides and phenolic compounds, which are determined by structural compatibility and the relative ratios of polysaccharides to phenolics (Lee et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In some cases, the gastrointestinal tract acts as a polyphenol extractor by breaking down solid food structures through the mechanical and chemical actions of the oral and digestive phases, respectively (Parada \u0026amp; Aguilera, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Tagliazucchi et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, depending on the overall composition of the food matrix, it could change. Other studies have observed a significant reduction in polyphenol recovery in the gut phase, likely due to interactions with dietary macronutrients such as dietary fiber, minerals, or proteins, as well as degradation during digestion, which can decrease their solubility, bioaccessibility, and efficacy (Correa-Betanzo et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; McDougall et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we hypothesized that the observed increase in TSPC during digestion is likely associated with the release of macromolecular or non-extractable polyphenols previously bound to the dietary fiber fraction of seaweeds, which plays a key role in limiting their initial extractability (P\u0026eacute;rez-Jim\u0026eacute;nez et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\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\u003eChanges in the total contents of soluble polyphenols (TSPC), phlorotannins (PhT), and antioxidant capacity (DPPH and ORAC methods) of seaweed-based matrices (CE, MS and SS) after \u003cem\u003ein vitro\u003c/em\u003e gastrointestinal digestion\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBrown seaweed-based matrices\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUndigested\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDigested\u003c/p\u003e\u003cp\u003e(Standard model)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDigested\u003c/p\u003e\u003cp\u003e(Older adult model)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eTSPC\u003c/p\u003e\u003cp\u003e(mg PhE /g dw)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e22.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e226.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003eaC\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e74.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003ebA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003ebC\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e50.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ecA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ecC\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003ecB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003ePhT\u003c/p\u003e\u003cp\u003e(mg PhE /g dw)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ebA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eDPPH\u003c/p\u003e\u003cp\u003e(umol T eq/g dw)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ebA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003csup\u003ecA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ecC\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eORAC\u003c/p\u003e\u003cp\u003e(umol T eq/g dw)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e101.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7\u003csup\u003ebA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e529\u0026thinsp;\u0026plusmn;\u0026thinsp;64\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e511\u0026thinsp;\u0026plusmn;\u0026thinsp;85\u003csup\u003eaB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e62.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003ecA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e217.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e482\u0026thinsp;\u0026plusmn;\u0026thinsp;75\u003csup\u003eaC\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e128.0\u0026thinsp;\u0026plusmn;\u0026thinsp;12.0\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e139.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003csup\u003ecA\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e165.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7\u003csup\u003ebB\u003c/sup\u003e\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 results represent the mean of three repetitions with their standard deviation. \u003csup\u003eA,b,c\u003c/sup\u003e Different lowercase letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between seaweed-based products (within the column). \u003csup\u003eA, B, C\u003c/sup\u003e Different capital letters indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between digestion models (within a row).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe initial PhT of the three seaweed-based matrices exhibited notable differences (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The CE showed the highest baseline concentration (0.8 mg PhE/g dw), followed by the MS with 0.2 mg PhE/g dw, and the SS, with a very low value (\u0026lt;\u0026thinsp;0.1 mg PhE/g dw). These variations are primarily attributed to the processing conditions, particularly temperature, with CE being the only matrix obtained through low-temperature USAE extraction at 40\u0026deg;C, which helps preserve phlorotannins. After \u003cem\u003ein vitro\u003c/em\u003e digestion, CE was the only matrix that maintained PhT bioaccessibility above 100%, with a recovery index of 130% under standard conditions and 112% in the older adult model. In the specific case of CE, the PhT values likely correspond to the phlorotannins previously identified in this extract, including carmalols, fucols, and eckols. The cleavage of their characteristic aryl\u0026ndash;ether and aryl\u0026ndash;aryl linkages under gastrointestinal conditions may lead to their release in more soluble forms, which could partly account for the higher PhT levels observed after digestion (Rivera-Tovar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, MS and SS exhibited a significant decrease (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e) in PhT, with much lower bioaccessibility values, ranging from 28% to 42% for MS and 36% to 51% for SS, regardless of the digestion model applied (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This suggests that matrix composition plays a key role in phlorotannin release, as MS and SS likely retained phlorotannins within more complex matrices that limited their extractability and digestion-driven release (Tarko \u0026amp; Duda-Chodak, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e gastrointestinal digestion study showed an antagonistic antioxidant effect in MS and SS matrices, attributed to the ability of marine polysaccharides to trap phenolic compounds. Correspondingly, previous research has highlighted that benzene rings and -OH groups from phlorotannins can non-covalently interact through hydrogen bonding and Van der Waals forces with the sulfate and hydroxyl groups of fucoidan, a primary polysaccharide in brown seaweed (Lee et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Liu \u0026amp; Le Bourvellec, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This effect becomes more pronounced in MS and SS due to their higher concentrations of polysaccharides and crude fibers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which influence the release of phenolic compounds under simulated gastrointestinal conditions. Phlorotannins are firmly bound to cellular and structural polysaccharides, such as fucoidans and alginates, that can form a three-dimensional gel-like network that captures phlorotannins (Liu \u0026amp; Le Bourvellec, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIntegrating all previous values and considering that the recommended serving sizes for these products differ significantly, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the differences in TSPC and PhT among the three \u003cem\u003eD. incurvata-\u003c/em\u003ebased products (CE, MS, and SS) based on recommended serving sizes. Notably, the CE, administered at 1 g of lyophilized extract, exhibited the highest initial TSPC (22 mg PhE) and PhT (0.7 mg PhE). In contrast, MS and SS, despite their larger serving sizes (1.6 g and 80 g, respectively), showed lower initial values for both TSPC and PhT. Following the \u003cem\u003ein vitro\u003c/em\u003e digestion, substantial differences were observed in the bioaccessible polyphenols, depending on the food matrix and digestive model. The serving size of CE displayed a remarkable increase in TSPC post-digestion, reaching 227 mg PhE under standard conditions and 75 mg PhE in the older adult model. Conversely, the MS and SS products exhibited lower polyphenol recoveries. However, a notable enhancement was observed for the serving size of SS under the standard model (405 mg PhE) due to its larger initial mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). CE consistently maintained higher values than MS and SS regarding phlorotannins, reflecting its high potential as a functional food ingredient.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Stability of the antioxidant capacity of seaweed-based matrices\u003c/h2\u003e\u003cp\u003eAn evaluation of antioxidant capacity complemented previous measurements of phlorotannin content as a first step in predicting their biological activities. The DPPH assay results (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed significant differences in antioxidant capacity among the undigested seaweed-based samples. MS presented the highest scavenging capacity, with a value of 42.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026micro;mol T eq/g dw, followed by CE at 15.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;mol T eq/g dw. SS exhibited the lowest DPPH scavenging capacity, with a 10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 \u0026micro;mol T eq/g dw. As previously observed for other vegetable-based matrices, DPPH scavenging capacities of CE, MS, and SS were not entirely associated with TSPC or PhT. This is likely because DPPH capacity depends on the molecular structure and reactivity of polyphenols rather than solely on their concentration (Tierney et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Processing methods, such as micronization or low-temperature USAE, can also influence antioxidant capacity by altering the accessibility and effectiveness of polyphenols in neutralizing free radicals. Thus, even with varying polyphenol concentrations, molecular structure and processing conditions contribute to differences in DPPH scavenging capacity (Rodr\u0026iacute;guez-Roque et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStandard digestion conditions significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the DPPH values of the undigested CE sample, increasing it from 15.6 \u0026micro;mol T eq/g to 26.8 \u0026micro;mol T eq/g dw (2-fold). However, digestion under older adult conditions did not cause an increase in this indicator (15.0 \u0026micro;mol T eq/g dw). Among the three seaweed-based matrices, undigested MS showed the highest DPPH scavenging capacity (42.7 \u0026micro;mol T eq/g dw), which decreased substantially following digestion under standard (2.5 \u0026micro;mol T eq/g dw) and older adult (1.9 \u0026micro;mol T eq/g dw) conditions. Similar to MS, DPPH values of SS significantly decreased 22-fold and 14-fold after digestion under standard (from 10.5 \u0026micro;mol T eq/g dw to 0.5 \u0026micro;mol T eq/g dw) and older adults (from 10.5 \u0026micro;mol T eq/g dw to 0.7 \u0026micro;mol T eq/g dw) simulated conditions, respectively. Nevertheless, the older adult digestion model had significantly less impact on the decrease in the SS DPPH scavenging capacity.\u003c/p\u003e\u003cp\u003eThe modifications in DPPH values were also expressed in terms of the recovery index to evaluate the stability of this \u003cem\u003ein vitro\u003c/em\u003e bioactivity descriptor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The DPPH recovery index of CE increased by up to 171% after \u003cem\u003ein vitro\u003c/em\u003e digestion under standard conditions, while 96% of its antioxidant capacity was retained in older adult samples. MS and SS presented a significant loss of their antioxidant capacity after digestion, maintaining 4% and 7% of the DPPH values, respectively, regardless of the digestion model applied (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eThe DPPH assay evaluates antioxidant capacity based on the ability of compounds to donate hydrogen atoms, a function strongly dependent on the presence and accessibility of hydroxyl (-OH) groups. In this study, CE presented the highest DPPH scavenging capacity after digestion, probably due to the presence of high-molecular-weight phlorotannin oligomers that, upon digestion, released smaller molecules with accessible hydroxyl groups. These smaller, more reactive molecules exhibited a greater interaction with the DPPH radical, resulting in a higher apparent antioxidant capacity (Prior et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This finding aligns with our previous study (Rivera-Tovar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), which identified the presence of high-molecular-weight phlorotannin oligomers in the \u003cem\u003eD. incurvata\u003c/em\u003e extract.\u003c/p\u003e\u003cp\u003eIn contrast, the MS and SS samples contained higher amounts of low-solubility carbohydrate oligomers, which likely formed interactions with phlorotannins, making them resistant to digestion (Parada et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This binding effect reduced the accessibility of hydroxyl groups, thereby hindering their ability to neutralize DPPH radicals. Consequently, even though these matrices may contain phenolic compounds, their radical-scavenging capacity was diminished due to steric hindrance and reduced availability of functional groups essential for the reaction. A similar trend to that observed for TSPC was found when comparing the DPPH values after the standard and older adult digestion models. This reduction is likely due to higher gastric pH and longer digestion times, which limit the release of polyphenols. These results highlight the impact of age-related digestive changes on the availability of antioxidants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConsidering the phlorotannins tentatively identified in the CE in a previous study (Rivera-Tovar et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), the antioxidant capacity was also assessed using the ORAC assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), which has been suggested to be more selective when the extract contains high-molecular-weight polyphenols (Rivera-Tovar et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). SS showed the highest antioxidant capacity, with a value of 128\u0026thinsp;\u0026plusmn;\u0026thinsp;12 \u0026micro;mol T eq/g dw, followed by CE with a moderate ORAC value of 101.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7 \u0026micro;mol T eq/g dw, while MS presented the lowest ORAC value at 62.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 \u0026micro;mol T eq/g dw (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe ORAC values for digested samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) showed that digestion markedly enhanced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the antioxidant capacity of CE, MS, and SS, with values of 101.0 \u0026micro;mol T eq/g dw, 62.6 \u0026micro;mol T eq/g dw, and 127.7 \u0026micro;mol T eq/g dw, respectively. This suggests that antioxidant compounds remain stable and effective after digestion, regardless of the digestive model employed. Under standard digestion conditions, the ORAC value of CE significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to 529.1 \u0026micro;mol T eq/g dw (5-fold), MS ORAC to 217.6 \u0026micro;mol T eq/g dw (3-fold), and SS ORAC to 139.2 \u0026micro;mol T eq/g dw (1.1-fold) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eThe digestive conditions model for older adults slightly decreased the ORAC value of CE, with no significant differences compared to the standard digestion model. Nevertheless, the bioaccessibility index of the antioxidant ORAC capacity of MS and SS was significantly enhanced (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), particularly in MS (up to 482.3 \u0026micro;mol T eq/g dw and 165.8 \u0026micro;mol T eq/g dw, respectively), which showed a value 8-fold higher than its undigested counterpart.\u003c/p\u003e\u003cp\u003eAccording to the ORAC results, the CE and MS were the matrices with the highest potential bioactivity, showing a substantial increase in their bioaccessibility indexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Under standard digestion conditions, the CE, MS, and SS showed increases in their bioaccessibility indexes of 524%, 347%, and 109%, respectively. Furthermore, the older adult digestion model appears to have a distinct impact on the ORAC bioaccessibility index, particularly for MS, where the bioaccessibility index is significantly higher (770%) than in the standard model. In contrast, SS remained the least effective in terms of the ORAC bioaccessibility index, although it improved under the older adult digestion model (130%). These results underscore the complex, non-linear relationship between the phlorotannin concentration in the three seaweed-based carriers and their antioxidant capacity.\u003c/p\u003e\u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis study highlights that the obtaining process, matrix composition, and age-related digestive changes are key determinants of the bioaccessibility of phlorotannins in brown seaweed-based products. The CE, which can be considered a safe and sustainable food ingredient with antioxidant capacity obtained through low-temperature USAE, showed higher phlorotannin stability and bioaccessibility compared to commercially available products obtained through higher-temperature processes (MS and SS). Furthermore, simulated digestive conditions for older adults revealed significant variations in phlorotannin bioaccessibility, likely due to age-related physiological changes such as reduced enzyme activity and altered gastric pH. The antioxidant capacity of seaweed-based products increased significantly following digestion; however, it remained unaffected by alterations related to the older adult model, thereby confirming that seaweed is a promising raw material for developing functional foods targeted at the elderly.\u003c/p\u003e\u003cp\u003eFuture studies should perform a more detailed analysis of the chemical and structural features of antioxidant compounds in brown seaweed, considering that other compounds with antioxidant capacity, such as sulfated carbohydrates, might also be present in USAE extracts. Furthermore, clinical studies are necessary to verify the bioavailability of brown seaweed phlorotannins in older adults, including the potential contribution of the colonic microbiota, as well as to evaluate the long-term health effects of seaweed-based products, particularly for aging populations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eETHICS DECLARATION\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eFUNDINGS\u003c/h2\u003e\u003cp\u003eANID-Chile funded this research through the FONDECYT Regular grants 1220097 and 1230115, FONDECYT Iniciaci\u0026oacute;n grant 11230112, and the FOVI grant 240263.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMAR: Conceptualization, data curation, figure preparation, and manuscript writing. GRA, LS: Experimental work and data analysis; LS also participated in manuscript editing. JPJ, SGSA, JRPC, OCV, MMC, FPB: Conceptualization and manuscript editing.MSMC: Conceptualization, writing, and editing of the manuscript. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmerican Public Health Association, American Water Works Association, \u0026amp; Water Environment Federation. (1999). \u003cem\u003eStandard methods for the examination of water and wastewater\u003c/em\u003e (American Public Health Association, Ed.).\u003c/li\u003e\n\u003cli\u003eAOAC International. (2012). \u003cem\u003eOfficial Methods of Analysis of AOAC International (19th ed.). Gaithersburg, MD, USA: AOAC International. Method 942.05.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eAOAC International. (2012). \u003cem\u003eOfficial Methods of Analysis of AOAC International (19th ed.). Gaithersburg, MD, USA: AOAC International. Method 986.25.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eAOAC International. 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In \u003cem\u003eTrends in Food Science and Technology\u003c/em\u003e (Vol. 143). Elsevier Ltd. https://doi.org/10.1016/j.tifs.2023.104301\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"seaweed, phlorotannin, antioxidant capacity, bioaccessibility, older adults","lastPublishedDoi":"10.21203/rs.3.rs-7427244/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7427244/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAddressing age-related health challenges through improved nutrition is increasingly important as the global population ages. Concurrently, sustainable food systems are focusing on low-impact ingredients, such as brown seaweed, which require minimal resources and offer promising health benefits. However, concerns about heavy metal accumulation in brown seaweed highlight the challenges associated with its safe use. After confirming the safety of a sustainable \u003cem\u003eDurvillaea incurvata\u003c/em\u003e extract, containing phlorotannins from trimers to octamers and mannitol, through an 80% reduction in heavy metals, this study evaluated the bioaccessibility of phlorotannins and the stability of antioxidant capacity during \u003cem\u003ein vitro\u003c/em\u003e gastrointestinal digestion in both standard and older adult models. It also compared these results with two commercially available seaweed products: micronized capsules and rehydrated salad. Crude extract exhibited the highest phlorotannin level before digestion for both Folin-Ciocalteu (22.5 mg PhE/g dw) and DMBA (0.8 mg PhE/g dw) assays. Phlorotannin bioaccessibility indices reached 1005% and 332% in the standard and older adult models, respectively, when total soluble polyphenol values were used for measurement. The DMBA assay confirmed an increase in phlorotannin content after digestion, likely due to the release of compounds previously bound to the food matrix; however, the differences observed between the two digestion models did not exhibit the same magnitude of change. The antioxidant capacity (DPPH assay) was highest in the micronized seaweed; however, it decreased after digestion. Conversely, the digested crude extract retained its antioxidant capacity under older adult conditions. These results support the use of brown seaweed extracts as safe and effective ingredients in foods for older adults.\u003c/p\u003e","manuscriptTitle":"Evaluation of phlorotannin bioaccessibility and antioxidant capacity stability of a safe and sustainable brown seaweed extract: role of age-related digestion changes and matrix composition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 16:00:15","doi":"10.21203/rs.3.rs-7427244/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":"f609a269-3b5b-4593-8d9a-b5eccbc72fc4","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:03:24+00:00","versionOfRecord":{"articleIdentity":"rs-7427244","link":"https://doi.org/10.1186/s43014-025-00359-4","journal":{"identity":"food-production-processing-and-nutrition","isVorOnly":false,"title":"Food Production, Processing and Nutrition"},"publishedOn":"2026-02-28 15:58:39","publishedOnDateReadable":"February 28th, 2026"},"versionCreatedAt":"2025-10-03 16:00:15","video":"","vorDoi":"10.1186/s43014-025-00359-4","vorDoiUrl":"https://doi.org/10.1186/s43014-025-00359-4","workflowStages":[]},"version":"v1","identity":"rs-7427244","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7427244","identity":"rs-7427244","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}