## 3 Results
### Nutritional Composition and Bioactivity  
#### 3.4.1 Nutritive Value  
The incorporation of different microalgae strains into bread resulted in significant changes in their nutritional composition (Table 6) compared to the control sample (\(p\) < 0.05). The microalgae significantly increased the bread's protein content, with _C. vulgaris_ biomass exhibiting the most pronounced increase (14.7%) in protein content compared to the other algae. In previous research, a significant increase in protein was observed when microalgae were added to baked products, which aligns with our results [1,7]. However, the protein content in the bread samples accounted for at least 12% of the energy value, suggesting that all bread samples, including the control, potentially meet the criteria for nutrition claims according to Regulation (EC) No. 1924/2006 [59]. In terms of lipids and ash, they also increased with the addition of microalgae. However, breads with _C. vulgaris_ and _T. chuii_ exhibited higher content in regard to both ash and lipid compared to _P. tricornutum_. A similar trend was found when _M. gaditana_ and _Chlamydomonas_ sp were added to gluten-free bread, resulting in an increase in the lipids and ash [29]. Breads containing _C. vulgaris_ and _P. tricornutum_ exhibited lower carbohydrate content than the control, while the addition of _T. chuii_ resulted in no significant variation in carbohydrate content. In general, it is important to indicate that the protein, lipid, and ash contents in the end products depend on the specific type of algae utilised and the conditions under which they are cultivated. In general, wheat grains typically contain low concentrations of calcium (Ca), potassium (K), and magnesium (Mg), which can be partially removed with the bran during the milling processes [60]. The addition of microalgae biomass can enhance these mineral contents, which are essential for bone health, nerve function, and maintaining overall well-being. Their presence in a diet is vital for preventing various health issues and supporting vital bodily functions [61]. The mineral profile of wheat bread improved when enriched with microalgae biomass, as shown in Table 7. Bread containing _C. vulgaris_ exhibited higher levels of iron (Fe) compared to the control (\(p\) < 0.05). In contrast, bread with _T. chuii_ and _P. tricornutum_ did not exhibit a significant difference (\(p\) > 0.05) (Table 7). It is worth noting that the \begin{table}
\begin{tabular}{l c c c c c} \hline \hline \multicolumn{1}{c}{\multirow{2}{*}{**Sample**}} & \multicolumn{1}{c}{**Moisture**} & \multicolumn{1}{c}{**Carbohydrates**} & \multicolumn{1}{c}{**Protein**} & \multicolumn{1}{c}{**Lipids**} & \multicolumn{1}{c}{**Ash**} & \multicolumn{1}{c}{**Energy**} \\  & **g/100 g** & **g/100 g** & **g/100 g** & **g/100 g** & **g/100 g** & **kJ/100 g** \\ \hline Control & 42.97 \(\pm\) 0.12 a & 44.75 \(\pm\) 0.06 a & 10.62 \(\pm\) 0.05 d & 0.03 \(\pm\) 0.00 d & 1.63 \(\pm\) 0.02 c & 221.62 \\ Cv & 42.75 \(\pm\) 0.15 a & 42.30 \(\pm\) 0.21 c & 12.18 \(\pm\) 0.02 a & 0.16 \(\pm\) 0.00 a & 2.61 \(\pm\) 0.06 a & 219.19 \\ Pt & 42.93 \(\pm\) 0.50 a & 43.87 \(\pm\) 0.47 b & 11.31 \(\pm\) 0.02 b & 0.04 \(\pm\) 0.00 c & 1.84 \(\pm\) 0.03 b & 221.23 \\ Tc & 41.47 \(\pm\) 0.12 b & 44.75 \(\pm\) 0.17 a & 11.08 \(\pm\) 0.03 c & 0.14 \(\pm\) 0.00 b & 2.56 \(\pm\) 0.04 a & 224.46 \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Centesimal composition and energy value of bread samples. Control: Without microalgae; _Chlorella vulgaris_ (Cv), _Phaeodactylum tricornutum_ (Pt), and _Tetraselmis chuii_ (Tc). Different letters in the same parameter show significant differences (\(p\) < 0.05, Tukey test). incorporation of microalgae increased the levels of potassium (K), calcium (Ca), magnesium (Mg), phosphorus (P), sulphur (S), and manganese (Mn). The addition of _T. chuii_ resulted in a substantial increase in both Ca and Mg levels, surpassing all other bread formulations. However, _P. tricornutum_ increased the K, Mg, S, and Mn in comparison to the other bread samples.