Influence of Alaria esculenta powder on sensory acceptance, nutritional aspects, and staling of wheat bread

Influence of Alaria esculenta powder on sensory acceptance, nutritional aspects, and staling of wheat bread Leonard Mihaly Cozmuta a,* , Anca Peter a , Camelia Nicula a , Alexandra Uivarasan a , Zsolt Szak´ acs a , Sonia Scarfi b,c,d , Maha Moussa b,d , Serena Mirata b,c , Paula Fajardo Bern´ ardez e , Sara Baldassari f , Giuliana Drava f , Anca Mihaly Cozmuta a a Technical University of Cluj Napoca, North University Center of Baia Mare, Victoriei Str. 76, Baia Mare, Romania b Department of Earth, Environment and Life Sciences, University of Genova, Corso Europa 26, 16132, Genova, Italy c Inter-University Centre for the Promotion of the 3Rs Principles in Teaching & Research, 10129 Turin, Italy d National Biodiversity Future Center (NBFC), 90133 Palermo, Italy e Biotechnology and Health Department, ANFACO-CYTMA, Colegio Universitario, Vigo, Spain f Department of Pharmacy, University of Genova, 16148, Genova, Italy ARTICLE INFO Keywords: Alaria esculenta bread Glycemic index Bread staling Functional bread ABSTRACT This study investigated the nutritional, sensory, physiological, and shelf-life impacts of incorporating Alaria esculenta seaweed powder into wheat bread. Structural analysis showed that high levels (7.5–10 %) reduced porosity and volume, whereas moderate inclusion (2.5–5 %) maintained favorable crumb properties. Sensory evaluation revealed overall acceptability scores of 8.15 for control bread (B-C) and 8.12 for bread with 5 % A. esculenta (B-5A). Proximate composition analysis showed increases in protein (1.26-fold), fiber (1.47-fold), and ash (5.63-fold) in B-5A, while energy value and carbohydrate contents decreased (1.48and 1.26-fold, respectively). In vitro assays using THP-1 macrophages and Caco-2 cells confirmed the absence of cytotoxicity up to 200 µg/mL, with slight viability enhancements. Inflammatory gene expression (IL6, IL8, MCP1, TNF α ) remained unchanged, with a modest IL6 reduction in Alaria-treated macrophages. In vivo glycemic testing showed moderated postprandial peaks for B-5A and B-C (≈130 mg/dL at 30 min) compared with glucose (146 mg/dL at 15 min), while B-5A maintained a more stable plateau between 90–120 min, suggesting prolonged satiety. During storage, B-5A exhibited superior moisture retention (28.12 % vs. 22.01 %), slower starch retrogradation (44.76 %→47.95 % vs. 45.27 %→50.29 %), and a more ordered protein structure, with greater increases in α -helix (+14.13 %) and reductions in random coil (–12.40 %). Protein surface hydrophobicity evolved more gradually in B-5A (contact angle decrease 40.48◦→35.51◦) than in B-C (66.86◦→50.92◦), reflecting enhanced water-binding. Collectively, these differences indicate slower staling and extended shelf life. Overall, 5 % A. esculenta fortification enhances nutritional quality, delays staling, improves shelf life, and maintains consumer acceptability without adverse cytotoxic or inflammatory effects. 1. Introduction White bread, a staple in various diets worldwide, has long been criticized for its limited nutritional value and its tendency to stale rapidly. While it remains a key food product in many cultures due to its convenience and affordability, its drawbacks have prompted growing concern regarding its role in human health. These concerns primarily focus on its composition, which is based on refined wheat flour, resulting in a lack of essential nutrients compared to whole-grain bread (Williams, 2012). A key issue with white bread is its high glycemic index, which is associated with a rapid increase in blood sugar levels upon consumption and a higher risk of developing conditions such as type 2 diabetes, cardiovascular diseases, and obesity (Lambadiari et al., 2020). Another significant issue with white bread is its prone for rapid staling, a cascade Abbreviations: AA score, amino acids score; B-C, control bread; B-2.5A, bread containing 2.5% Alaria esculenta reported to the weight of flour in control bread; B5A, bread containing 5% Alaria esculenta reported to the weight of flour in control bread; B-7.5A, bread containing 7.5% Alaria esculenta reported to the weight of flour in control bread; B-10A, bread containing 10% Alaria esculenta reported to the weight of flour in control bread. * Corresponding author. E-mail address: [email protected] (L. Mihaly Cozmuta). Contents lists available at ScienceDirect Future Foods journal homepage: www.elsevier.com/locate/fufo https://doi.org/10.1016/j.fufo.2025.100805 Received 2 September 2025; Received in revised form 21 October 2025; Accepted 21 October 2025 Future Foods 12 (2025) 100805 Available online 24 October 2025 2666-8335/© 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ). of processes that occur immediately after baking (Amigo et al., 2021). The staling process is driven by the retrogradation of starch, a phenomenon in which the amylopectin molecules in the starch of bread crystallize, leading to a loss of moisture and a subsequent deterioration in the texture and taste of the bread (Willhoft, 2007). Stale limits the shelf life of white bread, makes it less palatable, and more prone to spoilage, thus contributing to the economic and environmental burden associated with food waste (Bartek et al., 2025). In recent years, there has been growing interest in fortifying white bread with functional ingredients that can enhance its nutritional profile and improve its shelf life. Seaweeds are promising ingredients due to their rich content in bioactive compounds, including polysaccharides, proteins, vitamins (such as vitamins A, C, and B12), essential minerals (such as iodine, calcium, and magnesium), carotenoids, phenolic compounds and saponins (Senadheera et al., 2023). These bioactive components offer a range of potential health benefits, from supporting immune function to improving gut health and reducing the risk of chronic diseases (MacArtain et al., 2007). Alaria esculenta, a brown seaweed, is particularly notable for its high content of soluble fibers, antioxidants, and minerals (Lytou et al., 2021). Studies have shown that incorporating seaweed into food products can enhance their mineral content, provide prebiotic fibers that benefit gut health, and impart antioxidant properties (Matos et al., 2024). In bread, the addition of seaweed has been shown to improve the overall nutritional profile, increase fiber content, and reduce the glycemic index of the final product (Quitral et al., 2022). Furthermore, seaweed has been suggested to help mitigate staling due to its hydrophilic nature, which may assist in retaining moisture and prolonging shelf life (Kılınç et al., 2013). While A. esculenta offers these nutritional and functional benefits, it can also accumulate heavy metals or excessive iodine (Cherry et al., 2019), which may pose health risks if consumed in large amounts or over prolonged periods. Therefore, its elemental composition should be monitored to ensure safety when used in food applications. Despite the potential benefits of fortifying bread with seaweed, there is a noticeable gap in the scientific literature regarding the nutritional value of bread containing A. esculenta. While studies have examined the incorporation of seaweed into various food products (Roohinejad et al., 2017), few have specifically focused on A. esculenta and its effects when used in bread formulations. Moreover, the impact of A. esculenta on the staling process of white bread remains underexplored. Research on the relationship between the presence of A. esculenta and the mechanisms of bread staling, such as starch retrogradation and changes in protein structure, could provide valuable insights into how A. esculenta influences bread texture and shelf life. This could potentially lead to new functional food products that combine improved nutritional quality with extended freshness. In this regard, this study aims to explore the potential of fortifying white bread with A. esculenta to improve the nutritional profile of bread and potentially mitigate the rapid staling process. 2. Materials and methods 2.1. Raw materials and reagents White flour (Baneasa, Romania), dry yeast (Pakmaya, Romania), salt (Salrom, Romania), sunflower oil (Unisol, Romania) were purchased from a local supermarket. Alaria esculenta powder with granulometry <2 mm was supplied by The Seaweed Company (Ireland). The Technical Sheet provided by the supplier is presented in Table S1 from the Supplementary Material.. Reagents HNO 3 , HCl, NaOH, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid, acetone, Folin-Ciocalteu, Na 2 CO 3 , chloroform, gallic acid, pepsin, trypsin, chymotrypsin, trichloroacetic acid (TCA), 2 % ninhydrin solution, and l-glycine standards were purchased from Merck & Co., Inc. (Darmstadt, Germany). 2.2. Bread preparation The formulations of bread loaves were developed by partially substituting refined wheat flour with A. esculenta powder at different incorporation levels (2.5 %, 5.0 %, 7.5 %, and 10 % w/w related to the wheat flour content). The control formulation (B-C) consisted exclusively of 1000 g of wheat flour, whereas the experimental formulations (B-2.5A, B-5A, B-7.5A, and B-10A) contained 975, 950, 925, and 900 g of wheat flour, respectively, with to the addition of 25, 50, 75, and 100 g of A. esculenta powder, respectively. The amounts of dry yeast (14 g) and sunflower oil (30 g) were held constant across all formulations. The salt concentration, however, was gradually reduced from 15 g in control and 2.5 % substitution breads to 13 g, 12 g, and 10 g in the 5 %, 7.5 %, and 10 % formulations, respectively. This adjustment was based on preliminary sensory evaluations aimed at mitigating excessive saltiness attributable to the inherent mineral content of A. esculenta. The water addition varied with substitution level, ranging from 600 mL in the control dough to 640 mL in the 10 % substitution, with incremental increases of 10 mL per formulation. The optimal water weight was determined through preliminary hydration capacity tests. The ingredients were mixed for 15 min using a Biovita MB-1500 PRO (Cluj Napoca, Romania) at 2200 rpm until homogeneous doughs were formed. The doughs were then proofed for 1 hour in a fermentation chamber set to 30 ◦C and 75 % relative humidity (Matina, Bucharest, Romania). The doughs were shaped into 750 g loaves by hand and placed in baking trays (15 cm ×8 cm ×6 cm). They were allowed to rise for an additional 30 min at 30 ◦C and 75 % relative humidity. The loaves were baked in a preheated oven for 15 min at 200 ◦C, followed by 75 min at 180 ◦C. After baking, the loaves were removed from the trays and allowed to cool for 30 min at 25 ◦C before undergoing sensory evaluation. 2.3. Appearance of whole bread and their cross-sections Pictures of freshly prepared breads and selected bread samples during staling were captured using a Canon 80D digital single-lens reflex camera (Canon Inc., Japan). 2.4. Sensory evaluation The sensory analysis of the bread loaves was conducted at room temperature (22 ◦C) under daylight, with a group of 47 healthy panelists, of which 76.60 % were females and 23.40 % males, with ages between 18–50 years, who were habitual bread consumers. The study was approved by the Ethics Committee of the Technical University of ClujNapoca (Romania, Nr. 594/10.04.2024) and adhered to the ethical principles outlined in the 1975 Declaration of Helsinki, as revised in 2013. Before the sensory evaluation, the participants were informed about the study’s purpose and provided written consent. They were also trained to develop a consensus on the descriptive vocabulary for the bread samples. The samples of each bread formulation, cut into 10×5 cm pieces, were coded, randomly placed on plastic plates, and presented to panelists in a sequential monadic testing format using a complete block design. In addition, panelists were given whole loaves of bread and their cross-sections. They were asked to complete a questionnaire in which they rated the attributes of appearance, color, crust strength, crumb structure, elasticity, volume, taste, smell, and overall acceptability using a nine-point hedonic scale, where 1-extremely unpleasant; 2-strongly unpleasant; 3-unpleasant; 4-slightly unpleasant; 5-neutral; 6-slightly pleasant; 7-pleasant; 8-strongly pleasant; 9-extremely pleasant. To prevent the carryover effect, participants rinsed their mouths with room temperature (25 ◦C) water between tasting each bread formulation. The total score for each attribute was calculated as the average of the individual scores. The A. esculenta-based bread with the highest overall acceptability score, as well as the control bread, were selected for further investigations. L. Mihaly Cozmuta et al. Future Foods 12 (2025) 100805 2 2.5. Proximate analysis Proximate analysis of selected bread loaves in terms of moisture (AOAC 925.10, 1925), protein (AOAC 945.18-B, 1995, with a 6.25 nitrogen-to-protein conversion factor), fat (AOAC 920.39.C, 1995), ash (AOAC 936.07, 2013), and fiber (Commission Regulation No. 152/2009) was conducted according to standardized methods. The carbohydrate content was calculated using the equation: Carbohydrates(g/100g)=100− − (Moisture+Protein+Fat+Ash+Fiber) (1) Considering the energy supplied by carbohydrates, proteins, fiber, and fat, the energy provided by B-C and B-5A was calculated using the following formula: 2.6. Chlorophylls and carotenoids content Chlorophylls and carotenoids extraction was performed in acetone at a bread-to-acetone ratio of 10:1 (w:v). After 1 min, the solution was centrifuged for 15 min at 6000 x g. A volume of 15 μ L of the freshly prepared extract was diluted with 150 μ L of acetone, and the absorbance was spectrophotometric measured (PerkinElmer Aanalyst 800, SUA) at 662 nm, 645 nm, 470 nm, 653 nm, and 654 nm (Hynstova et al., 2018; Lichtenthaler and Buschmann, 2001). The results were calculated using Eqs. (3)–(8), and expressed as μ g/100 g of dry bread: Ca =11.24∗A662 −2.04∗A645 (3) Cb=20.13∗A645 −4.19 ∗A662 (4) Ca +b=7.05∗A662 +18.09 ∗A645 (5) Cx+c= (1000∗A470 −1.90 ∗Ca −63.14 ∗Cb)/214 (6) Ccc= (Ca+b) + (Cx +c)(7) Cph= (321.3∗A653) − (208.4∗A654)(8) where Ca is the concentration of chlorophyll a, μ g/mL; Cb is the concentration of chlorophyll b, μ g/mL; Ca+b is the concentration of total chlorophylls, μ g/mL; Cx+c is the concentration of total carotenoids, μ g/ mL; Ccc is the concentration of total chlorophyll and carotenoids, μ g/ mL; Cph is the concentration of total pheophytins, μ g/mL. 2.7. Cell cultures and A. esculenta bread extracts used in the in vitro tests For the safety assessment of human consumption of A. esculenta based bread, the potential toxicity of the B-5A was evaluated in vitro using several physiologically relevant cellular models simulating the human gastrointestinal tract. In this regard, the human intestinal Caco-2 cell line undergoes a spontaneous differentiation into a polarised cellular monolayer with functional features of small intestinal enterocytes, ultimately mimicking the human intestinal epithelial barrier (Ferruzza et al., 2012; Natoli et al., 2012). Similarly, THP-1-derived macrophages represent a valuable in vitro model to obtain a more in-depth understanding of the inflammatory processes carried out by resident intestinal macrophages in the gastrointestinal tract (Phuangbubpha et al., 2023). As a result, both cellular models have been extensively used for toxicological studies (Susewind et al., 2016; Hempt et al., 2020; Polet et al., 2020; Song et al., 2024; Giordani et al., 2025). Therefore, the cytotoxic potential of B-5A was evaluated on THP-1-derived M0 macrophages, on undifferentiated Caco-2 cells and on enterocyte-like Caco-2-derived monolayers at different time points (i. e., 24 and 72 h) using the MTT test. B-C was employed as a negative control. The human colon epithelial carcinoma cell line Caco-2 was obtained from the Interlab Cell Line Collection (IRCCS Ospedale Policlinico San Martino, Genoa, Italy) and cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM) (Biowest, Nuaill´ e, France) supplemented with 10 % FBS (Biowest), 4 mM l-glutamine (Corning Inc., NY, USA), 1 % non-essential amino acids (Corning Inc.), and penicillin/streptomycin (Corning Inc.) as antibiotics. For experimental procedures, Caco-2 cells were used either as undifferentiated cells or as differentiated enterocytelike monolayers to mimic the human intestinal epithelial barrier. Differentiation was achieved by seeding Caco-2 cells at 300,000 cells/cm² on Transwell® PET inserts (12 mm diameter, 0.4 μ m pore size) (VWR, Milan, Italy) with complete medium in both the apical and basolateral compartments. After the formation of a confluent monolayer, Caco-2 cells were cultured in complete medium for 21 days, with media changes performed three times a week (Ferruzza et al., 2012; Natoli et al., 2012) to support their differentiation. Both cell lines were cultured at 37 ◦C in a humidified atmosphere containing 5 % CO₂. The human monocytic cell line THP-1 was sourced from the American Type Culture Collection (LGC Standards srl, Milan, Italy) and maintained in RPMI-1640 medium supplemented with 2 mM l-glutamine (Euroclone, Milan, Italy) and 10 % fetal bovine serum (FBS) (Euroclone). For experimental purposes, THP-1 cells were differentiated into M0 macrophages by the addition of 30 ng/mL phorbol-12-myristate 13-acetate (PMA) (PeproTech EC, London, UK) to the culture medium for 24 hours (Di Giuseppe et al., 2022). To prepare the water-soluble bread (WSB) extracts for the biological tests, 10 g of dried bread samples (B-5A and B-C) were dissolved in 80 mL of deionized water and homogenised for 1 min with an ULTRATURRAX® T25 basic (IKA-Werke GmbH, Staufen, Germany) before sonication for 1 min at 100 Hz with an UP100H Ultrasonic Processor (Hielscher Ultrasonics GmbH, Teltow, Germany). After agitating for 30 min at room temperature with a magnetic stirrer, the WSB extracts were centrifuged at 4000 rpm for 45 min and passed through a filter paper to remove residual sunflower oil from the supernatant. Once collected, the extracts were centrifuged at 14,000 rpm for 10 min before the supernatant was sterilized using a 0.22 μ m syringe filter (VWR®, Milan, Italy). For the in vitro toxicological experiments, each solution was diluted directly in the culture media to obtain the different final concentrations as reported below. 2.8. Toxicity assessment with MTT test The potential cytotoxicity of B-5A WSB was assessed at 24 and 72 h in THP-1-M0 macrophages and in Caco-2 undifferentiated cells. For both time-points, Caco-2 cells were seeded at 15,000 cells/well in 96-well plates, while THP-1 monocytes were plated at 50,000 cells/well and induced to differentiate by treatment with PMA. Moreover, the potential toxicity of B-5A WSB was also evaluated in Caco-2 enterocyte-like monolayers, differentiated as described above. For all experiments, B-5A and B-C WSB were added to each well (200,100 and 50 μ g/mL final concentrations), and the plates were incubated at 37 ◦C for 24/72 h. Energyvalue(kcal/100g)= 4×Carbohydrate(%)+4×Protein(%)+2×Fiber(%)+9×Fat(%)(2) L. Mihaly Cozmuta et al. Future Foods 12 (2025) 100805 3 Then, cell viability was evaluated by the MTT assay (0.5 mg/mL final concentration) as reported by Pozzolini et al. (2016). Data are the means ±SD of two independent experiments in which each condition was tested eight times and results are expressed as percentage of cell viability respect to untreated control cells (C). 2.9. Inflammatory cytokine expression by qPCR To assess whether consumption of B-5A could somehow elicit inflammatory responses, the gene expression of several inflammatory mediators (i.e., IL-1β, IL-6, IL-8, MCP1, and TNFα ) was investigated in THP-1-M0 macrophages treated with B-5A and B-C WSB and the results were compared to those of LPS positive control, a well-known pro-inflammatory stimulus. THP-1 cells were plated at 500,000 cells/well in 6well plates and, once differentiated into M0 macrophages, cells were challenged with B-5A and B-C WSB (200 μ g/mL final concentration) for 24 h. Then, the gene expression profile of inflammatory mediators, interleukin-1β (IL-1β, a.n. NM_000576.3), interleukin-6 (IL-6, a.n. NM_001318095.2), interleukin-8 (IL-8, a.n. NM_000584.4), monocyte chemoattractant protein-1 (MCP1, NM_002982), and tumour necrosis factor-alpha (TNFα , a.n. NM_000594.4) was evaluated by qPCR relative to untreated control cells. Values were normalised to GAPDH (a.n. NM_002046) and HPRT-1 (a.n. NM_000194.3) mRNA expression. RNA extraction, cDNA retrotranscription and qPCR analyses were performed as described in Giordani et al. (2025). All primer pairs are reported in Table S2. 2.10. In vivo glycemic index The influence of consuming control bread and Alaria-bread on postprandial blood glucose levels was assessed in relation to raw glucose. The study was approved by the Ethics Committee of the Technical University of Cluj-Napoca, Romania (no. 594/10.04.2024). A total of 50 healthy subjects, aged 18 to 60 years, were included in the study. Individuals with health conditions were excluded. Before the experiment, subjects were informed about the objective of the study and provided their informed consent. The study was conducted following a minimum of 10 hours of fasting, across three different days, with a oneday break in between. The participants consumed 54.36 g of control bread (B-C), 80.50 g of Alaria-bread (B-5A), and 26.05 g of raw glucose (adjusted for the carbohydrate content in the bread) and drank 100 ml of water within 10 min. Blood glucose levels were measured using an AccuChek Active Glucometer (Roche Diabetes Care, Inc., Basel, Switzerland), with samples taken from the fingertip of each subject every 15 min over a two-hour period following the ingestion of the bread loaves and glucose. The experiment was repeated three times. 2.11. Staling of selected bread types Following cooling for 1 hour, the bread loaves were stored in clean polyethylene bags at 25 ◦C and 48 % relative humidity for 4 days. The staling process of the selected bread samples was examined with respect to the evolution of appearance, changes in moisture content, crust strength, specific volume, dispersion coefficient, color, changes in protein and starch structures. 2.11.1. Specific volume The rapeseed displacement method was used to determine the specific volume (V) of bread, calculated using Eq. (9): V(cm3/g)= (m1+m2−−m3)∗100/(m1∗d),(9) where m 1 is the weight of the bread sample, g; d is the density of rapeseeds, 1.02 g/cm³; m 2 is the weight of the container filled with rapeseeds, g; m 3 is the weight of the container with both the sample and rapeseeds, g. 2.11.2. Crust strength Crust strength was assessed using a Wagner FDK10 force dial penetrometer (Wagner Instruments, USA) on a 15-point scale at the minimum setting, and the average values were reported. 2.11.3. Dispersion coefficient in bread loaves The bread loaf was cut horizontally at its highest point, creating a cross-sectional surface. The height (H) and diameter (D) of the loaf were measured at five different positions using a caliper, and the H/D index was calculated based on the average values. 2.11.4. Image analysis The image analysis, conducted using ImageJ software (Schneider et al., 2012), investigated the evolution of bread crumb structure over a four-day staling period through solidity analysis. Solidity, a shape descriptor used to quantify the compactness of pores, was calculated as the ratio between the area of a pore and its convex area. Solidity values range from 0 to 1, where a value of 1 indicates a very compact, smooth (solid-like) pore, and lower values correspond to more irregular and open pores. The solidity values were grouped into nine categories (0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, and 1.0), and the number of pores (dimensionless) falling within each solidity range was plotted for Days 1 to 4. 2.11.5. Reflectance spectra The speciation of individual-colored compounds in food products is a time-consuming process that incurs high costs for both instrumentation and reagents. Reflectance spectra have been effectively employed to predict the concentrations of protein, amylose, total polyphenols, and fiber in rice, as well as its antioxidant activity (Xie et al., 2014). However, no literature has been found regarding the use of reflectance spectra to identify optically active compound groups and their co-pigmentation in bread fortified with seaweed. Such an approach could provide insights into the sensory properties of enriched bread, such as antioxidant activity, specific tastes in terms of astringency and bitterness. The color changes during the staling process of control and Alariaenriched bread were evaluated through the analysis of reflectance spectra. Reflectance spectra of the samples at various stages of staling were recorded over a wavelength range of 360 to 780 nm, with 10 repetitions at a 1 nm resolution. Data acquisition was performed using a UV/VIS Lambda 35 spectrometer (PerkinElmer, USA), coupled with a Labsphere RSA-PE-20 integrating sphere. The reflectance spectra were deconvoluted into seven discrete wavelength bands corresponding to defined regions of the visible spectrum: 380–449 nm (purple), 450–484 nm (blue), 485–499 nm (cyan), 500–564 nm (green), 565–589 nm (yellow), 590–624 nm (orange), and 625–760 nm (red). These spectral regions are indicative of optically active, color-contributing compounds present in the bread samples. Their distribution provides valuable information on pigment composition as well as potential chemical alterations related to formulation differences and staling process. 2.11.6. FTIR spectra The FTIR absorbance spectra of the bread samples were recorded using a Perkin Elmer BX2 spectrometer (SUA) equipped with an ATR Pike Miracle diamond crystal, covering a measurement range of 1550–6000 cm⁻¹. FTIR analysis was performed by placing the bread samples directly onto the crystal under the same pressure. Background spectra were recorded prior to sample measurement. The samples were analyzed without any prior preparation to preserve the native starch and protein structures, as processing could induce structural alterations. Spectra were collected at a resolution of 4 cm⁻¹ by accumulating 30 repetitions. Structural changes in starch and the secondary structure of proteins (amide I band) were analyzed in the ranges of 4000–6000 cm⁻¹ and 1550–1700 cm⁻¹, respectively. To achieve this, FTIR spectra were processed according to the method proposed by Mihaly Cozmuta et al. L. Mihaly Cozmuta et al. Future Foods 12 (2025) 100805 4 (2025) through normalization and deconvolution, using the Savitzky–Golay coefficients for a third-order polynomial with a 25-point window, assuming a Gaussian distribution for the minima of the second-order derivative. The deconvoluted curves were analyzed in terms of the relative crystallinity degree for starch (1047 cm⁻¹ and 995 cm⁻¹ for the crystalline form, and 1020 cm⁻¹ for the amorphous form) (Wang et al., 2020). For protein structure, the ratio of secondary structural features was assessed, including α -helix (dense ordered structure, 1663–1646 cm⁻¹), β-sheet (ordered structure, 1636–1615 cm⁻¹, 1700–1682 cm⁻¹), β-turn (relatively extended ordered structure, 1681–1664 cm⁻¹), and random coil (disordered structure, 1645–1637 cm⁻¹) (Zhao et al., 2021). 2.11.7. Surface hydrophobicity of bread protein isolate Protein extraction was performed following the method described by Joshi et al. (2011). Bread samples were mixed with distilled water at a ratio of 1:15 (w:v), and the pH of the solution was adjusted to 9.5 using a 1.0 M NaOH solution. The mixture was homogenized for one hour at room temperature while stirring at 750 rpm. After removal of the coarse solid phase, the solution was centrifuged at 6000 x g for 30 min. The pH of the supernatant was then adjusted to 4.5 using a 1.0 M HCl solution. Protein precipitation was allowed to occur at room temperature (25 ◦C) for 5 hours. Following a second centrifugation under the same conditions, the liquid phase was discarded, and the protein isolate was collected. It was washed with distilled water to remove soluble components, neutralized to pH 7 with 1.0 M HCl, and the protein suspension was decanted and filtered. The protein isolate was then dried at 35 ◦C for 24 hours. The evolution of surface hydrophobicity of the protein isolates during bread staling was discussed based on the contact angle of a water drop, measured using the sessile drop method (Zhao et al., 2021). The protein isolate was spread on a glass slide, and a 5 μ L drop of distilled water was placed on its surface. Images were captured using a Canon EF 100 mm f/2.8 L Macro IS USM lens, set to its highest magnification (1x) and a high depth of field (f/13). Focus was adjusted by positioning the sample on a microscope sample holder. No additional white balance or image post-processing was applied, aside from the automatic settings applied by the camera. The contact angles were measured using ImageJ (Schneider et al., 2012) software. The contact angle was recorded on both sides of each drop, with measurements taken within 15 s to prevent water adsorption by the pellet. 2.12. Statistical analysis The experiments were conducted in triplicates, and the results are presented as the mean ±standard deviation. Contact angle and crust strength measurements were taken from at least 10 replicates, and the results are also expressed as mean ±standard deviation. To assess significant differences between group means, one-way ANOVA followed by Tukey’s test (GraphPad Software, Inc., San Diego, CA, USA), results with p values <0.01 or p <0.05 were considered significant. Principal Component Analysis (PCA) was performed using Statistica 7.0 (StatSoft Inc., Tulsa, USA) to highlight the underlying patterns and relationships between the bread formulation and staling parameters, enabling the identification of key factors driving variability and their contributions to the overall data structure. 3. Results and discussion 3.1. Appearance of bread varieties A comprehensive analysis of the structural and textural attributes of the formulated breads is presented in Table 1. Based on these findings, it can be concluded that the incorporation of low concentrations (2.5 %–5 %) of Alaria esculenta powder results in breads with a favorable balance between structural integrity and nutritional enhancement. Higher inclusion levels (7.5 %–10 %) alter the crumb texture, reducing porosity and making the bread denser. Although A. esculenta contributes to a darker crumb color and may enhance the nutritional value of bread, excessive incorporation negatively impacts key quality parameters such as texture, elasticity, and specific volume, factors that are likely to influence consumer acceptability. 3.2. Sensory study The results of the sensory evaluation of bread samples containing varying percentages of Alaria esculenta powder (B-C, B-2.5A, B-5A, B7.5A, B-10A) are presented in Figure S1. Concerning appearance, the control bread (B-C) received the highest score, indicating that its visual characteristics were most appealing to consumers. As A. esculenta powder was incorporated, a gradual decline in appearance scores was noted, particularly in B-7.5A and B-10A. The darker coloration and potential speckling due to the seaweed powder may have contributed to lower acceptability. However, B-2.5A and B-5A maintained favorable visual appeal, suggesting that moderate levels of A. esculenta powder do not significantly impact the appearance of the bread. Aroma scores declined as the percentage of Alaria esculenta powder increased. B-C had the highest aroma acceptability, whereas B-10A received the lowest scores. The introduction of seaweed powder imparted marine-like notes, which may not have met the consumer expectations for bread. B-2.5A and B-5A maintained a more neutral aroma profile, suggesting that at these concentrations, the impact on aroma is minimal. The texture scores followed a similar downward trend in bread with increasing A. esculenta content. While B-C had the most favorable texture rating, B2.5A and B-5A remained within an acceptable range. However, B-7.5A and B-10A showed significant declines, likely due to changes in crumb structure and moisture retention caused by the seaweed powder. Higher concentrations of A. esculenta may have affected the elasticity and softness of the bread, making it less palatable. Taste scores reflected a decline in consumer preference with increasing A. esculenta content. Also in this case, B-C had the highest rating, while B-10A exhibited the lowest. The presence of umami and marine flavors in higher concentrations may have contributed to the lower scores. However, B-2.5A and B-5A remained acceptable, indicating that small amounts of A. esculenta powder can be incorporated without drastically altering the taste profile. The overall acceptability scores reveal an interesting trend. While the control bread, B-C, received the highest rating (8.15), the B-5A sample (8.12) closely followed, indicating that a 5 % A. esculenta powder inclusion does not significantly impact consumer preference. The B-2.5A sample (7.74) was slightly lower but still within an acceptable range. These results suggest that moderate incorporation of A. esculenta powder (up to 5 %) does not drastically reduce acceptability and may even be comparable to the control. However, higher inclusion levels (B-7.5A and B-10A) introduced stronger sensory changes that led to lower overall scores. The sensory study indicates that the inclusion of A. esculenta powder affects consumer acceptability across multiple sensory parameters. Higher concentrations (B-7.5A and B-10A) led to a decline in preference, while lower levels (B-2.5A and B-5A) maintained an acceptable sensory profile. Notably, the B-5A sample exhibited an overall acceptability score very close to control, B-C, suggesting that it may be a promising formulation. The sensory study conducted by J¨ onsson et al. (2024) on wheat bread containing 7.5 % Alaria esculenta (relative to wheat flour), involving 49 consumers (45 % women and 55 % men), reported the following scores for Alaria-based bread: 6.5 for appearance, 5.9 for smell, 5.7 for taste, 6.2 for texture, and 5.9 for overall. In comparison, the corresponding scores for the control bread were 6.2, 6.2, 6.1, 6.5, and 6.3, respectively. Apart from appearance, where the bread with 7 % Alaria esculenta received a higher score than the control bread, the smell, taste, texture, and overall acceptability were rated with lower scores. For the attributes of appearance, smell, taste, and texture, the scores received by the bread containing 7.5 % A. esculenta in our study are similar to those reported L. Mihaly Cozmuta et al. Future Foods 12 (2025) 100805 5 Table 1 Structural and textural characteristics of the investigated breads. Feature Control Bread (B-C) Bread with 2.5% Alaria esculenta (B-2.5A) Bread with 5% Alaria esculenta (B-5A) Bread with 7.5% Alaria esculenta (B7.5A) Bread with 10% Alaria esculenta (B-10A)  Color Crumb: Bright white Crust: Goldenbrown Crumb: Light beige, minor greenish tint Crust: Golden-brown with slight dullness Crumb: Darker beige with green speckles Crust: Light brown, slightly rough Crumb: Brown-green, uniform darkening Crust: Thicker, rougher, darker brown Crumb: Deep brown-green, almost grayish Crust: Dark, rigid, cracked Crumb density Light, fluffy, well-risen Slightly denser, still soft Noticeably denser, slightly reduced expansion Much denser, signs of collapse Extremely dense, crumbly, weak internal structure Porosity Highly porous, fine, uniform distribution of pores Slightly reduced porosity, small and even pores Moderate porosity, smaller pores, slightly compacted Irregular porosity, uneven bubble distribution Low porosity, large voids and collapse areas Elasticity & spring High elasticity, rebounds well Still retains elasticity, minor firmness increase Moderate elasticity, firmer structure Reduced elasticity, crumb becomes rigid Low elasticity, crumb breaks easily Moisture retention Balanced moisture, soft and fresh Slightly drier but acceptable Noticeably firmer, minor dryness Much lower moisture, firmer and drier crumb Dry, brittle, lower water retention Gluten network strength Strong, well-formed gluten strands supporting structure Mostly intact, minor weakening Slight gluten weakening, causing reduced rise Significant gluten network weakening, less rise & structure Weak gluten network, leads to structural collapse Pores size and distribution Small to medium, evenly distributed Mostly small and uniform Smaller, numerous, but consistent Irregular, large voids in some areas Large, inconsistent, collapse-prone air pockets Crumb texture Smooth, light, well-structured Slightly firmer but smooth Denser, somewhat rougher Rougher crumb, fibrous mouthfeel Extremely rough, fibrous, brittle Crust thickness Thin, crisp crust with soft interior Slightly thicker crust, remains crisp Thicker crust, slightly firmer Noticeably thicker, harder crust Very thick, rigid crust, large cracks visible Structural integrity Strong, holds shape well when sliced Maintains shape, minor firmness increase Holds shape but denser Some deformation, weakened structure Weak structure, crumbles and collapses when sliced Loaf expansion Well-risen, airy, symmetrical loaf Good expansion, minor height reduction Reduced height, firmer crumb Clear loss of rise, slightly sunken Structural collapse, compacted loaf Aroma Traditional wheat aroma, bright white crumb Mild seaweed hints, slight beige hue Stronger earthy aroma, visible green flecks More pronounced earthy/seaweed scent, darker tone Strong seaweed aroma, deep dark crumb L. Mihaly Cozmuta et al. Future Foods 12 (2025) 100805 6 by J¨ onsson et al. (2024) for bread containing 7 % A. esculenta. However, for the overall acceptability attribute, our study reported a slightly higher score of 6.46 compared to 5.9 in the study of J¨ onsson et al. (2024). The similar scores rated for the bread containing A. esculenta by consumers from two distinct countries suggest that its sensory attributes, such as appearance, smell, taste, texture, and overall acceptability, were perceived similarly across these groups. This could indicate that the inclusion of A. esculenta in bread may offer a sensory profile that is acceptable in different cultural contexts, providing initial evidence for its potential in broader markets. Based on the results of the sensory study, the bread containing 5 % A. esculenta powder (B-5A) and the bread without the seaweed powder (B-C) were selected for further investigation. 3.3. Proximate composition The proximate composition of the selected breads is displayed in Table 2. The moisture content of B-5A is significantly higher than that of B-C, due to the water-binding properties of A. esculenta (Bennett et al., 2023), which could result in moister bread. Increased moisture can improve the softness and mouthfeel of the bread; however, it may also impact its shelf life by promoting microbial growth if not properly controlled. The protein content in B-5A is significantly higher compared to the control bread. The incorporation of A. esculenta, which is a seaweed known for its high protein content (Table S1, Bennett et al., 2023), clearly contributes to this increase. The higher protein content could also enhance the functional properties of the bread, such as improving its structure and texture. Both breads exhibit low-fat content, with no significant difference between the values. This suggests that the addition of A. esculenta powder does not alter the fat profile of the bread, reflecting the intrinsic composition of the seaweed, which is not a significant source of fat (Table S1). A significant increase in fiber content was observed in B-5A.A. esculenta is known for its high fiber content (Table S1), which contributes to improved digestive health and may help regulate blood sugar levels. The higher fiber content in B-5A may also enhance satiety, which could be beneficial for weight management. The ash content in B-5A is significantly higher than in B-C, indicating a higher mineral percentage. Given the typical recommended daily intake (RDI) for each nutrient in adults, in terms of 114 g/day protein for men and 102 g/day protein for women, 25 g/day fiber, 130 g/day carbohydrates, 70 g/day fat, and 2000 kcal/day energy (EFSA, 2017) the contribution of the selected breads to the RDI was calculated (Table 2). B-5A provides a higher percentage of the daily protein requirement compared to B-C, making it a more protein-rich option. B-5A also offers more fiber than B-C, contributing to a significantly higher percentage of the daily recommended intake, which could be particularly beneficial for the digestive system. On the other hand, the control bread, B-C, provides a greater percentage of the daily carbohydrate requirement, while B-5A contains fewer carbohydrates, which may appeal to low carbohydrate diets. Both breads are endowed with very small amounts of fat relative to the daily recommended intake (around 1 %). The energetic value of B-5A is lower than that of the control bread, reflecting the reduction in carbohydrate content. The lower caloric density of B-5A may be advantageous for consumers aiming to reduce caloric intake while benefiting from higher protein, fiber, and mineral content. However, this reduction in energy may also influence the texture and mouthfeel, potentially making the seaweed-based bread denser. The differences between the investigated breads highlight the potential benefits of incorporating Alaria esculenta into bread products, particularly for consumers seeking enhanced fiber, protein, and mineral intake, while also reducing carbohydrate and caloric consumption. 3.4. Citotoxicity study and inflammatory cytokine expression For consistency with the composition of the fluids that the bread would encounter in the digestive tract if consumed, the aqueous extracts (WSB) of the control bread, B-C, and of the 5 % A. esculenta-enriched bread, B-5A, were obtained and then tested in in vitro toxicity and inflammatory response studies. No significant differences were observed in the total phenolic content (~2.1 μ g/mg extract) and in the DPPH antioxidant scavenging activity (~21 % at 2.5 mg/mL extract) of the two B-C and B-5A breads (work protocols are provided in the Supplementary Material), indicating a similar composition of the compounds extracted in the two breads. Concerning the MTT cytotoxicity tests, in differentiated THP-1 M0 macrophages, at the 24-hour time point (Fig. 1A), all extracts, regardless of bread type or concentration, maintained cell viabilities at or above 100 %, indicating no harmful effects. At the highest concentration (200 µg/mL), a slight increase in cell viability was observed for both bread types, with values modestly exceeding that of the control, untreated cells. At 100 and 50 µg/mL, viability remained stable, with WSB from the B-5A Alaria-enriched bread consistently exhibiting slightly higher mean values compared to control bread extracts. Overall, no significant cytotoxicity was detected after 24 hours of exposure. The presence of A. esculenta powder did not adversely affect macrophage viability and may even confer a modest viability-enhancing effect at lower concentrations. After 72 hours of exposure (Fig. 1B), a similar trend of high cell Table 2 Proximate composition of control bread and bread containing 5 % Alaria esculenta powder. Parameter RDI, g/day (EFSA, 2017) Control bread, B-C Bread with 5% Alaria, B-5A Amount Contribution to the RDI/100g of bread Amount Contribution to the RDI /100g of bread Moisture, g/100 g37.25 ±0.60 b - 41.32 ±0.70 a - Protein, g/100 g (dry sample) 114 for men 102 for women 10.12 ±0.04 b 8.88 % for men 9.92 % for women 12.73 ±0.08 a 11.17 % for men 12.48 % for women Fat, g/100 g (dry sample) 70 0.85 ±0.04 a 1.21 % 0.72 ±0.02 a 1.03 % Fiber, g/100 g (dry sample) 25 2.40 ±0.00 b 9.60 % 3.53 ±0.00 a 14.12 % Ash, g/100 g (dry sample) - 1.66 ±0.01 b - 9.34 ±0.03 a - Carbohydrates, g/100 (dry sample) 130 47.92 ±0.88 a 36.86 % 32.36 ±0.54 b 24.89 % Energetic value, Kcal/100 g (dry sample) 2000 243.81 ± 4.75 a 12.19 % 193.90 ± 3.70 b 9.70 % RDI –recommended daily intake; The contributions to the RDI were calculated based on EFSA (2017) guidelines for adult males and females aged 19–50 years. Individual requirements may vary based on age, sex, health status, and physiological conditions. Data are presented as mean ±standard deviation (n =3). Values with different letters a and b in the same line are significantly different (p <0.05). Values for protein, fat, fiber, ash, carbohydrates and energetic value are reported to the dry sample. L. Mihaly Cozmuta et al. Future Foods 12 (2025) 100805 7 (caption on next page) L. Mihaly Cozmuta et al. Future Foods 12 (2025) 100805 8 viability persisted. A slight increase above control levels was again observed for both extracts, B-C and B-5A WSB, particularly at 200 µg/ mL, where the B-5A WSB appeared to promote marginally greater viability than the B-C. At 100 µg/mL and 50 µg/mL, cell viability remained at high level. While the differences were minor, the B-5A WSB consistently demonstrated a slight elevation in viability relative to the control. Comparable outcomes were obtained in undifferentiated Caco-2 cells (Fig. 1C and D). Across all concentrations tested (up to 200 µg/mL), both B-C and B-5A WSB were non-cytotoxic. The inclusion of 5 % A. esculenta powder did not negatively impact cell viability and, in some cases, may have slightly improved it, particularly at the 24-hour time point (Fig. 1C). Upon prolonged exposure of 72 hours (Fig. 1D), viability trends remained stable or slightly increased, suggesting no accumulation of cytotoxic effects over time. The minimal variability between 24 h and 72 h data further supports the biocompatibility of both extract types. These findings suggest that the incorporation of Alaria esculenta into bread does not compromise intestinal epithelial cell viability and may help sustain cellular homeostasis. Similarly, no cytotoxicity was observed in differentiated Caco-2 enterocyte-like monolayers following 24 and 72 hours of treatment with the extracts. At 24 hours (Fig. 1E), cell viability remained consistently high across all extract types, with no significant reductions observed. Interestingly, after 72 hours (Fig. 1F), the B-5A WSB at 200 µg/mL appeared to induce a slight increase in metabolic activity, with viability approximately 8.1 % higher than that of control, untreated cells. These findings confirm that A. esculenta powder inclusion in bread does not compromise enterocyte-like Caco-2 cell welfare even after extended exposure. Notably, while the B-C WSB alone may slightly enhance cell proliferation at higher concentrations over 72 hours, the presence of A. esculenta in the B-5A bread appears to regulate this effect, maintaining cell viability near baseline levels. This may suggest a modulatory influence of A. esculenta on cell proliferation, without exerting cytotoxic stress. Our results align with the findings of O’Sullivan et al. (2011), who reported that methanolic extracts of brown seaweeds (e.g., Ascophyllum nodosum, Laminaria hyperborea, Pelvetia canaliculata, Fucus vesiculosus, and Fucus serratus) did not compromise Caco-2 cell viability at concentrations up to 2 mg/mL, as determined using the MTT assay. Also, the study of Huwait et al. (2022) which indicated that fucoidan, an algal-derived polysaccharide, expressed no cytotoxicity on THP-1 macrophages, even at 200 µg/mL for 24 h. Concerning the inflammatory potential in vitro study, as expected, exposure to lipopolysaccharide (LPS) positive control resulted in a significant transcriptional upregulation of IL-6, IL-8, MCP-1, and TNFα in THP-1 macrophages after 24 hours, with fold increases of 2.8, 1.5, 1.8, and 1.6, respectively, compared to untreated controls (Fig. 1G). In contrast, treatment with either B-C or B-5A WSB did not elicit significant alterations in the expression of these inflammatory mediators. However, a slight reduction in IL-6 mRNA levels was observed in macrophages treated with the B-5A WSB. Also, no significant differences in cytokine expression were detected between the two bread WSB treatments. Overall, the in vitro toxicological findings support the safety of A. esculenta-enriched bread, as neither toxic nor pro-inflammatory effects were observed in the examined intestinal (Caco-2) and immune (THP-1 macrophage) cell models. Bioactive compounds commonly found in brown seaweeds, including fucoidan, phlorotannins, fucoxanthin, and sterols, have been reported to modulate oxidative stressrelated signaling pathways, enhance cellular defense mechanisms, and exhibit therapeutic potential in the management of inflammationassociated disorders (Begum et al., 2021). Specifically, fucoidan extracts derived from species such as Undaria pinnatifida, Fucus vesiculosus, Ascophyllum nodosum, Laminaria japonica, and Macrocystis pyrifera have been shown to suppress the secretion of TNFα , IL-1β, and IL-6 in human peripheral blood mononuclear cells (PBMCs) and THP-1 macrophages following LPS stimulation (Ahmad et al., 2021). Furthermore, in THP-1-derived macrophages, fucoidan has been demonstrated to attenuate the release of pro-inflammatory cytokines and inhibit cell migration (Li et al., 2017). Future investigations, by means of organotypic in vitro models of intestinal inflammation, will help in highlighting the above-mentioned possible functional effects of A. esculenta enriched functional foods such as the bread designed in our study. 3.5. In vivo glycemic index Fig. 2 shows the postprandial blood glucose response within 120 min after the consumption of raw glucose, control bread (B-C), and bread with 5 % Alaria esculenta (B-5A), with standard deviations of values ranging between 6.73 % and 36.80 %. The ANOVA analysis of results (Table S3) did not indicate significant (p <0.05) differences due to high data variability. Statistically validated conclusions were obtained by using the data between the 25 % and 75 % quartiles corresponding to the best results in the middle range of variation, with standard deviations ranging between 1.64 % and 12.87 % (Table S4). The profile for raw glucose indicates a rapid increase, with the highest blood glucose level of 146.00 mg/dL reached after 15 min. After peaking, blood glucose dropped sharply up to 45 min to 105.6 mg/dL and then continued declining steadily, reaching nearly baseline at 89.6 mg/dL after 120 min. A moderate rise in blood glucose was observed for the control bread, with a maximum of 130.5 mg/dL after 30 min, followed by a gradual decline to near-baseline level at 96.2 mg/dL after 120 min. A similar trend was also observed for A. esculenta-bread, B-5A, with no significant differences in the maximum value or the time required to reach it compared to the control bread. The decline was gradual but slightly slower than in B-C, with final blood glucose level of 94.00 mg/dL, not significantly different from that of B-C. The comparative analysis of the evolution of blood glucose responses indicates that, after the consumption of raw glucose, the glucose peak in the blood is reached faster (15 min) and at a higher level (146.00 mg/dL) compared to the consumption of B-C and B-5A (30 min, 130.50 mg/dL and 133.80 mg/dL, respectively). In the case of B-5A, the evolution of blood glucose level was not significantly different from that of control bread during the 90 min, but it remained at a higher value than B-C during the 90–120 min range. It suggests that bread fortified with 5 % A. esculenta not only controls the magnitude of the postprandial glycemic response relative to raw glucose ingestion but also influences the temporal profile of blood glucose levels compared to control bread (B-C). While both bread samples show a lower glycemic peak than raw glucose, B-5A maintained a more stable blood glucose plateau ranging between 90and 120-min post-consumption, whereas B-C showed a continued decline toward baseline values. This prolonged maintenance of blood glucose could result in several metabolic advantages. A more sustained glycemic Fig. 1. In vitro cytotoxicity assessment of B-C and B-5A WSB extracts. Viability of THP-1-M0 macrophages treated with B-C and B-5A WSB extracts, after 24 h (A) and 72 h (B) by the MTT test. Viability of undifferentiated Caco-2 cells measured after 24 h (C) and 72 h (D) in the same conditions as (A). Viability of enterocyte-like Caco-2-derived monolayers measured after 24 h (E) and 72 h (F) in the same conditions as (A). Results are expressed as cell percentages relative to untreated control cells (C) and are the mean ±SD of two independent experiments in which each condition was tested eight times. Asterisks indicate significance in a paired Tukey test vs. C (** p <0.0001). G. Gene expression of pro-inflammatory mediators by qPCR. Gene expression analysis of IL-1β, IL-6, IL-8, MCP1 and TNFα in THP-1-M0 macrophages treated for 24 h with WSB B-C and B-5A at 200 µg/mL final concentrations. The positive control with LPS was at 500 ng/mL final concentration. Data are normalised to the GAPDH and HPRT housekeeping genes. Results are expressed as mRNA fold increase compared to control cells (C) and are the mean ±SD of three experiments performed in triplicate. Asterisks indicate significance in Tukey test vs C (ANOVA: IL-1β p <0.05, IL-6 p <0.000001, IL-8 p <0.005, MCP1 p < 0.000001, TNF p <0.0005; Tukey vs. C, ** p <0.005). L. Mihaly Cozmuta et al. Future Foods 12 (2025) 100805 9 intestinal organotypic in vitro models as well as formulation adjustments, such as masking undesirable flavors and optimizing texture, to enhance the palatability of A. esculenta-enriched bread, thereby allowing the incorporation of higher amounts of A. esculenta to achieve improved beneficial properties of the bread. Although no cytotoxic effects were observed in bread containing Alaria esculenta, future studies should quantitatively assess the levels of heavy metals and iodine in both the seaweed and final product. Such analyses would help determine whether consumption remains within safe limits and ensures that longterm intake does not pose health risks. Ethical statement The sensory study presented in the manuscript was approved by the Ethics Committee of the Technical University of Cluj-Napoca (Romania, Nr. 594/10.04.2024) and adhered to the ethical principles outlined in the 1975 Declaration of Helsinki, as revised in 2013. Before the sensory evaluation, the participants were informed about the study’s purpose and provided written consent. We confirm that this manuscript has not been published elsewhere in whole or in part and is not under consideration by any other journal. Approval of all Authors’ institutions have been granted to publish this work. All authors have approved the manuscript and agree with its submission to Future Foods. Funding sources This study has received funding from the European Union’s Horizon Europe research and innovation funding programme under research project Demonstration of innovative functional food production systems based on a more sustainable value chain of marine and freshwater raw materials for consciencious European consumersNOVAFOODIES, Programm HORIZON.2.6 - Food, Bioeconomy Natural Resources, Agriculture and Environment, HORIZON.2.6.4 - Seas, Oceans and Inland Waters; Topic: HORIZON-CL6-2022-FARM2FORK-02-05-two-stageInnovative food from marine and freshwater ecosystems. Grant Agreement No 101084180. doi:10.3030/101084180. Disclaimer Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. CRediT authorship contribution statement Leonard Mihaly Cozmuta: Writing – original draft, Software, Investigation, Conceptualization. Anca Peter: Investigation. Camelia Nicula: Investigation. Alexandra Uivarasan: Investigation. Zsolt Szak´ acs: Investigation. Sonia Scarfi: Methodology, Investigation. Maha Moussa: Methodology, Investigation. Serena Mirata: Methodology, Investigation. Paula Fajardo Bern´ ardez: Methodology, Investigation. Sara Baldassari: Methodology, Investigation. Giuliana Drava: Methodology, Investigation. Anca Mihaly Cozmuta: Writing – review & editing, Project administration, Methodology, Investigation. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study. Acknowledgements We would like to express our sincere gratitude to The Seaweeds Company, Ireland, for providing us with Alaria esculenta powder for this study. 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