**4. Discussion**

Seaweeds are a rich source of nutrients, but they are also an important source of different kinds of bioactive substances, including sulphated polysaccharides, carotenoid pigments, and phlorotannins, with potential health benefits. In particular, phlorotannins, derived primarily from brown algae, have

been recently found to possess multiple physiological activities, such as antioxidant, antibacterial, and anti-inflammatory [37].

Moreover, seaweeds provide health benefits due to biological effects attributed to some polyand/or oligosaccharides. These include prebiotic effects, immunomodulation, affecting the ability of white blood cells to attack tumor cells, reduction of the symptoms of respiratory tract infections, and protection against infectious diseases [30].

The link between the tissue oxidative damage caused by the increase of intracellular content of ROS and several pathophysiological conditions, such as aging, obesity, non-alcoholic fatty liver disease (NAFLD), type 2 diabetes mellitus, and cognitive decline, is highlighted by many preclinical and clinical studies [38–40]. However, an intracellular basal content of ROS, physiologically generated from both normal mitochondria and peroxisomes metabolism and different cytosolic enzyme systems, is important for some physiological processes. Therefore, the maintenance of intracellular redox homeostasis is fundamental. It depends on efficient antioxidant systems that provide a decrease of ROS production and scavenging free radicals. This antioxidant system includes antioxidants and antioxidant enzymes, such as vitamin E, vitamin C, glutathione, superoxide dismutase, glutathione peroxidase, and catalase [41].

Moreover, oxidative stress is also associated with lipid accumulation and peroxidation. High ROS levels induce the production of lipid peroxyl radicals, which can generate other extremely reactive products as malonyldialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), and 4-hydroxy-2-hexenal (4-HHE), typically used as markers of lipid oxidation [42]. Since the Western diet notably involves increased fat intake, thereby resulting in oxidative stress and impaired inflammation status, antioxidant compounds are frequently added into food.

In the last decades, the food industry has been focused in replacing synthetic antioxidants with natural compounds; of current interest are those related to bioactive compounds extracted from the edible seaweeds. Among these marine algae, *F. vesiculosus* is the most well-known species from the *Fucus* genus [25], representing an abundant and widely distributed kind of brown, perennial and edible seaweed. It is present in the cold-temperate waters of the northern hemisphere and can counteract oxidative stress. In fact, several studies have shown that it may act as an antioxidant either by direct scavenging ROS or stimulating the activity of endogenous antioxidant enzyme system [43]. Moreover, some of these studies confirmed the antioxidant effects of seaweed extracts, related to their significant polyphenol content.

The phlorotannins represent the major polyphenolics present in brown seaweed. These compounds are a subgroup of tannins, which are formed by the polymerization of phloroglucinol units. *F. vesciculosus* contains low molecular weight (LMW) phlorotannins between 4 and 8 phloroglucinol units, but also phlorotannins highly polymerized of up to 16 phloroglucinol units. In fact, Iceland colleagues have isolated in *F. vesiculosus* extracts both oligomeric and polymeric phlorotannins that have more antioxidant activity than monomeric compounds.

In our study, biochemical assays have confirmed that *F. vesiculosus* extracts, produced for healthy snack supplementation, have efficient antioxidant properties and a high content of polyphenols.

Moreover, both seaweed extracts, B200314 and B290814, prevent ROS formation in cell-based assays. Indeed, catalase and GSH reductase activities, measured in HepG2 cells, pre-incubated with seaweed extract B200314, do not increase in stress condition induced by TBUT exposure.

Other antioxidant mechanisms are rarely evaluated and discussed. In our study, we have observed the seaweed extracts capability to chelate Fe2<sup>+</sup> ions, and only one report from the literature [44] until now has suggested that seaweeds are metal-chelating agents, removing the metals and reducing their redox potential.

Essential heavy metals are cofactors in several biological processes: Cu, Zn, Fe, and Co are involved in oxygen utilization, cell growth, enzymatic reactions, biomolecular metabolism, and immunity system. Essential heavy metals homeostasis is wisely regulated through protein transporters responsible for their uptake, distribution, storage, and excretion. Metal accumulation in the human body leads to

damage to many organs, especially the nervous, respiratory, and reproductive systems [45]. In fact, metal accumulation induces ROS production, which results in membrane lipid peroxidation. Moreover, Fe and Cu ions catalyze hydroxyl radical's formation via Fenton-like reactions and react with DNA and proteins, resulting in their functional impairment. Finally, radicals can impact on mitochondria electron transport and metal excess in the cytoplasm can modify intracellular redox equilibrium, changing pH and protein conformation, which can lead to cellular dysfunction and apoptosis/necrosis.

Seaweeds, with their multiple polyphenols and oligo/polysaccharides enriched in a hydroxyl group and carbonyl group on ring C, have several sites for metal complexation able to chelate metal ions. Thus, *F. vesiculosus* may be considered a good chelating agen<sup>t</sup> because it forms chemically inert and non-toxic complexes with metal ions.

This study was part of the VII PQ European project, EnRichMar, aimed not only at screening of the bioactivity of seaweed extracts, but also at increasing the value of convenience foods by adding functional ingredients, produced from underutilized marine based raw materials and by-products (waste) from fish processing, with confirmed bioavailability. The focus has been placed on ingredients such as powder of fish oil and seaweed extracts, which may enhance positive health e ffects and stability, enhance flavor, and, consequently, contribute to salt reduction of the products to meet market demand.

Our results indicate that seaweed extract can be used in convenience food to increase their stability and antioxidant potential. Data also show the ability of flavorings to enhance with their polyphenol composition the e ffects of *F. vesiculosus* extracts [23]. The functional properties of the enriched products have been studied also via dietary intervention.

The results collected in the EnRichMar project, and partially illustrated in this study, also sugges<sup>t</sup> that rye snacks containing seaweed extracts show a higher polyphenol content and maintain antioxidant activity despite roasting process.

In conclusion, our preliminary approach sugges<sup>t</sup> that seaweed-based ingredients are potential natural antioxidants that could be used as active ingredients in functional food products as well as improving oxidative stability of healthy food products for targeted consumers.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/3/249/s1, Table S1. Overview of time and temperature during the production of the rye snack: Mixing, extrusion, drying, cooling, packaging, Table S2. List of raw materials, mix of flour, and extruded rye snacks utilized to study the antioxidant action of di fferent flavorings and di fferent amounts of seaweed extracts, Table S3. List of snack prototypes produced to study the effect of different flavorings and roasting on antioxidant properties of rye snacks enriched with *Fucus vesiculosus* extracts.

**Author Contributions:** The authors' responsibilities were as follows: R.J., K.S. and A.M.R., coordinated the analyses. G.M., S.Z., B.I. and P.A.C., conducted the experiments. A.M.R. and P.A.C. performed statistical analysis and interpreted the data. P.A.C., I.C. and A.M.R., wrote the manuscript. P.A.C., I.C., K.S., R.J. and A.M.R. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the European Community VII PQ SME-2013 Increased value of convenience foods by enrichment with marine based raw materials "ENRICHMAR" contract n. 606023.

**Acknowledgments:** The authors would like to acknowledge all the participant to the Enrichmar Consortium, in particular: the SMEs: BioActive Foods AS, Finnsnack/Ruislandia, Grímur kokkur ehf, Marinox ehf, Den Eelder; the RTD participants: Nederlandse Organisatie Voor Toegepast Natuurwetenschappelijk Onderzoek TNO, Teknologian Tutkimuskeskus VTT.

**Conflicts of Interest:** The authors declare no conflict of interest.
