The study aims to evaluate the bioavailability of phlorotannins and carotenoids from the seaweed Himanthalia elongata in healthy individuals after the consumption of a cracker fortified with this plant species. The study consists of an acute, blinded, randomized, controlled, crossover trial in which each participant consumes both the fortified cracker and a control cracker without seaweed. This design allows the comparison of the bioavailability of these compounds when incorporated into a bakery-based food matrix.

The BIO-ALGA study is a nutritional intervention designed to evaluate the bioavailability in humans of bioactive compounds present in the brown seaweed Himanthalia elongata, specifically phlorotannins and carotenoids. These compounds have attracted growing interest in the fields of nutrition and functional foods due to their potential antioxidant, anti-inflammatory, and metabolic regulatory properties. Marine macroalgae, and particularly Himanthalia elongata, are recognized as a valuable source of these compounds, in addition to providing dietary fiber, minerals, and other nutrients that may contribute to the prevention of chronic diseases such as obesity, type 2 diabetes, and cardiovascular disorders. However, for these compounds to exert physiological effects in the human body, they must be absorbed during digestion and reach the systemic circulation. Therefore, studying their bioavailability under real consumption conditions in humans is essential.

To address this objective, the study is designed as an acute, randomized, controlled, crossover, and blinded nutritional intervention conducted in healthy adults. Each participant will consume four functional cracker (approx. 20 g) on separate days: one enriched with Himanthalia elongata and another control cracker with identical formulation but without seaweed. The two interventions will be separated by a washout period of at least fifteen days to avoid carryover effects. The expected sample size is at least ten participants, ideally twelve, with balanced representation of men and women. Eligible participants will be healthy adults aged between 18 and 45 years with a body mass index between 18 and 30 kg/m². Exclusion criteria include, digestive diseases, allergies to the ingredients used in the study, current medication or supplement use, hormonal treatments, recent antibiotic consumption, or pregnancy.

The test product consists of four functional cracker weighing approximately 20 g and containing 1 g of freeze-dried Himanthalia elongata powder. This amount ensures the presence of the target bioactive compounds while remaining within safe dietary limits for iodine intake. The formulation includes rice flour, cornstarch, wheat flour, sesame, baking powder, egg and water. A little olive oil was used to enhance the absorption of carotenoids, such as fucoxanthin. Mannitol is used as a sweetener to provide sweetness without substantially increasing caloric content. The placebo bar has the same composition but does not contain seaweed, allowing direct comparison of the absorption of bioactive compounds delivered through the food matrix.

During the seven days prior to each intervention and throughout the study period, participants will be instructed to avoid the consumption of seaweed products. Additionally, they will complete a three-day dietary record before each intervention. On the intervention day, volunteers will attend the Human Nutrition Unit of the Institute of Food Science, Technology and Nutrition (ICTAN-CSIC) after an overnight fast of at least ten hours. A venous cannula will be inserted to allow repeated blood sampling, after which participants will consume the assigned cracker and remain at the research facility for the entire study day under the supervision of the research team. Throughout the day they will receive a standardized polyphenol-free diet to minimize interference with the analytical detection of metabolites derived from the seaweed.

Blood samples will be collected before intake and at multiple postprandial time points (0.5, 1, 1.5, 2, 2.5, 4, 6, 8, 19, and 24 hours) in order to evaluate the plasma kinetics of the absorbed compounds and their metabolites. Approximately 9 mL of blood will be collected at each time point, resulting in a total volume of 99 mL per intervention. Plasma will be separated by centrifugation and stored at -80 °C until analysis. In parallel, urine samples will be collected at defined intervals (-2 to 0 h, 0-3 h, 3-6 h, 6-9 h, and 9-24 h) to assess the urinary excretion of metabolites derived from the bioactive compounds present in the seaweed.

The collected biological samples will be analyzed using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry with electrospray ionization (HPLC-ESI-QTOF). Targeted metabolomic approaches will be applied to identify and quantify metabolites derived from phlorotannins and carotenoids present in Himanthalia elongata. Based on plasma concentration profiles and urinary excretion patterns, nutritional kinetic parameters such as maximum concentration (Cmax), time to reach maximum concentration (Tmax), and the area under the concentration-time curve (AUC) will be calculated, as well as the proportion of excreted metabolites relative to the ingested dose. This analytical strategy will allow the characterization of the real bioavailability of these compounds under physiological conditions and the identification of potential biomarkers of seaweed consumption.

The study poses minimal risk to participants, mainly limited to mild and transient discomfort associated with blood sampling. The ingredients used in the functional crackers have been carefully selected to ensure food safety and to maintain micronutrient intake, including iodine, within recommended limits. Although the study does not have a direct therapeutic objective, its results are expected to expand current knowledge about the metabolism and bioavailability of seaweed-derived bioactive compounds in humans and to provide scientific evidence supporting the development of functional foods incorporating marine algae with potential health benefits.

Inclusion Criteria:

• Body mass index: 18-30 Kg/m2

Exclusion Criteria:

• Following a vegetarian diet
• Being pregnant
• Suffering from digestive diseases (such as irritable bowel syndrome, gastrointestinal ulcers, etc.)
• Having a history of hypersensitivity or allergies to components of the study foods
• Following any medication regimen
• Currently taking or having recently taken antibiotics
• Having donated blood within the three months prior to the study

Individual participant data will be available in open data repositories and may be accessed once the study results are published.