**3. Results**

## *3.1. Bioactive Properties of Fucus Vesiculosus Extracts*

The antioxidant potential and the potential mechanism(s) of antioxidant action of seaweed extracts were characterized by a multiple-method approach, which include well-documented chemical assays and cell-based bioassays.

## 3.1.1. Total Polyphenol Content and Antioxidant Activity

The evaluation of total polyphenol content (TPC) is a reference assay largely used to measure polyphenols in foods. The TPC of the two batches of seaweed extracts (B200314 and B290814) was 0.26 and 0.30 g PGE/g extract, respectively (Table 1). The secondary metabolite composition of the *F. vesiculosus* was already studied in our laboratories, by HPLC-DAD-ESI-MS [21]. In accordance with the literature, we have described several phlorotannin compounds in seaweed extracts. In particular, we have detected phlorotannin tetramers, whose proposed structures were fucodiphlorethol A, and hexamer compounds at *m*/*z* 729, 622/621, and at *m*/*z* 462, whose proposed structures were trifucodiplorethol isomers. The lack of standards represents a limitation of the analytical method for these compounds.

The antioxidant capacity of the seaweed extracts was assessed by mean of ORAC, DPPH radical scavenging activity, and ferrous ion-chelating ability. The oxygen radical absorbance capacity expressed by ORAC value, a reference to compare the antioxidant value of foods, was 1545 μmol TE/g extract in B200314 and 1840 μmol TE/g extract in B290814 (Table 1). DPPH IC50 values of the extracts were 0.614 mg/mL for B200314 and 0.608 mg/mL for B290814 (Table 1). The ability of the extracts, B200314 and B290814, to chelate transition metal ions, especially Fe2<sup>+</sup> and Cu2+, demonstrated that their ferrous ion chelating ability (FCA) was higher (59% and 53%) at a concentration of 10 mg/mL, respectively (Table 1).


**Table 1.** Bioactive properties of the *Fucus vesiculosus* extracts assessed by di fferent chemical assays.

TPC, total polyphenol content; ORAC, oxygen radical absorbance capacity; DPPH. 2,2-DiPhenyl-1-PicrylHydrazyl radical scavenging activity, FCA, Fe2<sup>+</sup>-chelating activity. 1 g PhloroGlucinol Equivalents/g extract; 2 μmol Trolox Equivalents/g extract; 3 mg extract/mL.

#### 3.1.2. Cellular Antioxidant Activity of the *Fucus Vesiculosus* Extracts

To obtain a better prediction of the antioxidant activity of seaweed extracts we measured their ability to prevent the radical formation by CAA assay using HepG2 cells. The results, presented in Figure 1, indicate that both seaweed extracts were able *in vitro* to scavenge 50–60% of the AAPH-induced radicals. The extracts were active at very low concentrations (62.5 μg/mL and 31.3 μg/mL for B200314 and B290814, respectively); Trolox (50 μM = 12.5 μg/mL), used as a positive control, exhibited 98% cellular antioxidant activity.

**Figure 1.** Cellular antioxidant activity (CAA) in HepG2 cells as a % of the control. The control consisted of cells with the DCFH-DA probe and the AAPH peroxyl radical initiator, but in the absence of samples; the blank consisted of cells exposed to only the DCFH-DA probe. The values shown are the mean ± SD of three independent experiments.

#### 3.1.3. Cell Protective E ffects of Seaweed Extract against Induced Oxidative Stress

To assess the preventive capacity of seaweed extracts to protect cells from oxidative stress we assayed the activities of the major antioxidant enzymes in HepG2 cells pre-treated with seaweed extract and then stimulated with TBUT. Cells were exposed to 62.5 μg/mL B200314, the batch that was further implemented in the rye-snacks.

The results, shown in Figure 2, demonstrate that at this concentration, the seaweed extract displayed a protective e ffect on hepatic cells. In fact, the pretreatment with seaweed (Sw) extract determines a significant reduction of GSH reductase activity induced by TBUT exposure (Sw + TBUT vs. TBUT *p* < 0.05). Moreover, we observed a slight decrease of catalase activity induced by TBUT stress in cells pretreated with seaweeds, although this was not statistically significant. GSH peroxidase activity and total glutathione content remained to the level of stressed cells. No e ffects were observed in malonyldialdehyde (MDA) levels, the typical marker of lipid peroxidation, which were slightly increased by TBUT treatment.

**Figure 2.** Protective effects of seaweed extract assessed in HepG2 cells exposed to oxidative stress (TBUT) with or without 62.5 μg/mL seaweed extract (Sw). The main antioxidant enzymes are represented together with glutathione (GSH) and malonyldialdehyde (MDA) cell content. Values are expressed as mean ± SD of three independent experiments. \* vs. Ctrl (\* *p* < 0.05, \*\*\* *p* < 0.001); § vs. Sw (§§: *p* < 0.01, §§§: *p* < 0.001) and # vs. TBUT (#: *p* < 0.05, ###: *p* < 0.001).

#### 3.1.4. Development and Test of Enriched Rye Snacks

To test the ability of *F. vesiculosus* to increase snack antioxidant power, several rye snack samples enriched with various amounts of seaweed extract B200314 were produced by an optimized extrusion process (Table S1). This study included a rye snack model, as it was: (1) suitable due to uniformity of product; (2) interesting with regard to production process/heat treatment; and (3) interesting for its improved nutritional profile (iodine) and stability (potential antioxidant activity).

Several rye snack samples enriched with various amounts of seaweed extract were produced by extrusion, and the extrusion process was optimized. In spite of their health promoting impact, seaweeds cause an o ff-flavor to some extent in rye snacks. Thus, several experiments aiming to mask the o ff-flavor perceived in the snacks were also conducted.

To this aim, sensorial tests were performed and numerous snack samples were screened by a trained sensory expert panel (*n* = 5) by scoring and/or go/no go assessments. Promising samples were assessed by VTT's trained sensory panel. The test method used in sensory assessments was descriptive profiling. VTT's trained sensory panel (*n* = 10 or *n* = 2 × 10, i.e., duplicate session of the 10-member panel) evaluated the intensities of the sensory attributes on a linear scale 0–10, which was verbally anchored from both ends (0 = the attribute not perceived, 10 = the attribute very clearly perceived). In addition, the panelists gave verbal descriptions on the samples. The Compusense software collected data.

From these analyses, suitable snack options for the study were found. The most e ffective flavorings were garlic (G), basil+tomato (B/T), and rosemary (R). To increase the palatability of the snacks, roasting was also utilized. The development of prototypes of extruded rye snacks enriched with seaweed powder for production by Ruislandia were performed in parallel. The list of the produced and analyzed snacks, roasted and unroasted, is reported in Tables S2 and S3.

The antioxidant activity of the prototype snacks was firstly analyzed by chemical assays, taking into account the single components (raw materials) and their mixtures (mix of flour) before and after the extrusion process.

As shown in Figure 3, an appreciable increase of antioxidant activity, correlated to the increase of seaweed concentration, is evident in both the mix of flour and extruded snacks, even if the extract displays less activity compared to the same concentration of pure seaweed extract, showed in the raw material panel.

 **Figure 3.** *Cont.*

**Figure 3.** Trolox equivalent antioxidant capacity (TEAC) values for raw materials, mix of flour and extruded snacks with (**A**) 1% Garlic (G), (**B**) 0.5% Basil (B) + 3% Tomato (T), and (**C**) 0.5% Rosemary (R) powders and increasing concentration of seaweed extract (Sw). Ctrl consists in rye mix. Data are expressed as mean ± SD of three independent experiments.

Interestingly, the different flavorings, when added to mix of flour, showed an increment of TEAC value also in the absence of seaweed. Additionally, flavored extruded samples, without seaweed, showed an antioxidant potential in particular for rosemary and basil + tomatoes. Moreover, in a mix of flour and extruded snacks with different concentrations of seaweed, the antioxidant activity was similar, suggesting that the extrusion process did not modified the biological activity.

As regard to the palatability of the snacks, the 2% seaweed-enriched snack was considered the more interesting product, with a TEAC above 10 μmol/g sample.

In the final development of the products, extruded rye snacks were produced with 2.1% seaweed extract, corresponding to a theoretical 2500 ORAC value per portion (60 g), and flavored with garlic, basil+tomato, and rosemary, in roasted and unroasted conditions.

On these prototypes (Supplemental Table S3), we have evaluated the total polyphenol content and the oxygen radical absorbance capacity (Figure 4). The control snacks contained only 0.8% Himalayan salt as all other prototypes.

**Figure 4.** (**A**) TPC and (**B**) ORAC values (μmol TE in 100 g sample) of roasted (R) and unroasted (U) rye snacks containing different flavorings, i.e., 1% garlic (G), 0.5% basil (B) + 3% tomato (T) or 0.5% rosemary (R) powder. All groups contained rye mix, 0.8% Himalayan salt and 2.1% seaweed extract (Sw, batch B200314), except the control group, which only contained rye mix and 0.8% Himalayan salt (Ctrl). Data are expressed as mean ± SD of four independent experiments. \* *p* < 0.05 vs. Sw-U and Sw-R groups.

The addition of basil (0.5%) + tomato powder (3%) (B/T) or rosemary (0.5%) (R) to the seaweed enriched rye snack resulted in significantly (*p* < 0.05) higher TPC compared to other groups (Figure 4A). Similar trend was evident in the ORAC measures (Figure 4B) with significantly higher values in rye snacks containing the same flavorings. No changes were observed for different roasting processes.
