## 3 Results and Discussion
### Cellular Immunomodulatory Activity  
The activation of macrophages is an important part of initiating defensive reactions ensuring effective innate and adaptive immunity. As observed in Figure 1, exposure of RAW264.7 cells to the endotoxin LPS for 24 h upregulated generation of the inflammatory mediator NO, compared with the untreated control (\(p<0.0001\)). Figure 1 also demonstrates that all BWSPHs and SGID BWSPHs (0.5% (\(w/v\) dw)) increased NO production in LPS-induced RAW264.7 cells compared with the LPS control (\(p<0.05\)). The NO producing ability of BW-SPH-F was the only hydrolysate which was significantly reduced by SGID (\(p<0.05\)). Upon exposure to invasive species, pathogen-recognition receptors (PRRs) of immune cells stimulate numerous signalling cascades resulting in the upregulation of inflammatory cytokines such as TNF-\(\alpha\) and IL-6. TNF-\(\alpha\) controls inflammatory response through upregulation of proinflammatory cytokines IL-6 and IL-1\(\beta\) as well as through the upregulation of endothelial cell adhesion molecules inducing leukocyte extravasation [50; 51]. IL-6 plays an important role in immunity through terminal differentiation of B cells into immunoglobulin-secreting cells as well as regulating the balance between regulatory T cells and Th (T helper) 17 cells [52; 53]. No signal for IL-6 or TNF-\(\alpha\) protein expression was detected in untreated controls indicating DMEM and FBS were endotoxin free (data not shown). In addition to increasing NO production, hydrolysates BW-SPH-A, -B, -C and -F also increased IL-6 production in LPS-stimulated cells (\(p<0.05\)), with hydrolysates BW-SPH-C, -D, and E increasing levels of TNF-\(\alpha\) compared with the LPS control (\(p<0.05\)). This immunostimulation is likely to be lost as the hydrolysates transit the gut, as SGID data show that BW-SPH-F-GI was the only digest to enhance IL-6 and TNF-\(\alpha\) cytokine levels in LPS-activated macrophage (\(p<0.05\)) (Figures 2 and 3) compared with the LPS control, indicating the release of immunostimulant peptides from BW-SPH-F during the digestion process. Arithmetical ranking demonstrated that BW-SPH-F and BW-SPH-F-GI were the highest-ranking hydrolysates of the BWSPHs and SGID BWSPHs tested with respect to individual immunomodulatory parameters (Supplementary Table S4). No relationship between free amino acid content [26] and the immunomodulatory effect of BW-SPH-F-GI was observed, suggesting a role for peptides in its bioactivity. Figure 1: Effects of blue whiting soluble protein hydrolyses (BWSPHs) (BW-SPH-A–F) and simulated gastrointestinal digested (SGID) BWSPHs (BW-SPH-A–GI—F-GI) at 0.5% (\(w/v\) dry weight) on nitric oxide (NO) production in lipopolysaccharideaccharide (LPS)-challenged RAW264.7 mouse macrophage. RAW264.7 cells (1 \(\times\) 10\({}^{5}\) cells/mL, 200 \(\mu\)L/well) were simultaneously exposed to 2 \(\mu\)g/mL LPS and BWSPHs or SGID BWSPHs for 24 h. NO was measured using Griess assay and values were expressed as a percentage relative to positive control (Con), cells treated with 2 \(\mu\)g/mL LPS alone (100% NO secretion). Values represent the mean \(\pm\) SEM of three independent experiments. Significance was measured using ANOVA followed by Dunnett’s test. *, **, and **** denote statistically significant differences between sample and LPS control at \(p<0.05\), \(p<0.01\), and \(p<0.0001\), respectively. # indicates a significant difference at \(p<0.05\) between pre- and post-SGID values measured by \(t\)-test. Figure 2: The effect of blue whiting soluble protein hydrolyses (BWSPHs) (BW-SPH-A–F) and simulated gastrointestinal digested (SGID) BWSPHs (BW-SPH-A–GI—F-GI) at 0.5% (\(w/v\) dry weight) on IL-6 production in lipopolysaccharideaccharide (LPS)-stimulated RAW 264.7 cells. RAW264.7 cells (0.2 \(\times\) 10\({}^{5}\) cells/mL, 200 \(\mu\)L/well) were simultaneously exposed to 0.1 \(\mu\)g/mL LPS and BWSPHs or SGID BWSPHs for 24 h. Values were expressed as a percentage relative to positive control, cells treated with 0.1 \(\mu\)g/mL LPS alone (100% IL-6 secretion).