Assessment of blue whiting protein hydrolysate bioactivities using cell culture models
University College Cork
Low-value underutilised blue whiting fish represents a potential profitable source of protein for the generation of high-value, health-enhancing fractions. The research described in this thesis assessed the bioactive potential of blue whiting soluble protein hydrolysates (BWSPHs) using cellular model systems. Minced, deboned blue whiting was initially hydrolysed at laboratory scale to develop a protocol for the reproducible generation of eleven different BWSPHs. Cellular bioactivity analysis of these eleven hydrolysates indicated that none of the BWSPHs tested exhibited satiating activity, antioxidant activity, immunomodulatory activity or anti-obesity activity as measured using specific cellular models. However, six BWSPHs did exhibit anti-diabetic activity in vitro, therefore, these six BWSPHs were prepared at semi-pilot scale and all of the above bioactivities were reassessed in greater detail. The antioxidant and immunomodulatory activities of the six industrial-scale BWSPHs before simulated gastrointestinal digestion (SGID) (BW-SPH-A to BW-SPH-F) and after SGID (BW-SPH-A-GI to BW-SPH-F-GI) were assessed in stimulated murine RAW264.7 macrophage. Hydrolysate BW-SPH-A (0.5%, w/v dw), both pre- and post-SGID, increased the endogenous antioxidant, reduced glutathione (GSH), in tert-butylhydroperoxide (tBOOH)-treated cells and reduced reactive oxygen species (ROS) in H2O2-challenged RAW264.7 cells compared with stimulated controls (p<0.05). In vitro digested hydrolysate BW-SPH-F-GI (0.5%, w/v dw) induced immunostimulating effects in lipopolysaccharide (LPS)-activated RAW264.7 macrophages though increasing pro-inflammatory cytokine interleukin (IL)-6 and tumour necrosis factor (TNF)-α levels compared with the LPS-stimulated control (p<0.05). The satiating potential of the six BWSPHs was then assessed in murine enteroendocrine STC-1 cells. The ability of BWSPHs and SGID BWSPHs to modulate the secretion and/or production of satiety hormones glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK) and peptide YY (PYY) in STC-1 cells was studied as well as the signalling pathway activated by BWSPHs to modulate the secretion of these hormones. All BWSPHs (BW-SPH-A to BW-SPH-F) (1.0%, w/v dw) increased active GLP-1 secretion and proglucagon production in STC-1 cells compared to the basal control (Krebs-Ringer buffer) (p<0.05), possibly via intracellular calcium signalling, however this activity was lost following SGID. In addition, neither pre- nor post-SGID hydrolysates affected epithelial barrier integrity or stimulated IL-6 secretion in differentiated Caco-2/HT-29MTX co-cultured cells. The anti-obesity effects of BWSPHs and SGID BWSPHs was investigated using the murine 3T3-L1 cell line. Before SGID, hydrolysates BW-SPH-A, -B, -C and -F (1.0%, w/v dw) reduced triglyceride accumulation during preadipocyte differentiation (p<0.05), however none of the hydrolysates hydrolysed triglycerides in fully mature adipocytes. Interestingly, after SGID, all hydrolysates reduced triglyceride accumulation during differentiation and all except one BWSPH increased glycerol levels in mature adipocytes compared with the differentiated controls (p<0.05). Two anti-adipogenic hydrolysates, BW-SPH-A and BW-SPH-F, and their corresponding in vitro digests were observed to modulate triglyceride accumulation during preadipocyte differentiation via down-regulating the expression of key adipogenic transcription factors (peroxisome proliferator activated receptor (PPAR)-γ and CAAT (controlled amino acid therapy)/ enhancer binding protein (C/EBP)-α) compared with the differentiated controls (p<0.05). These SGID hydrolysates also exhibited anti-obesity activities following simulated intestinal permeation. After a 4 h exposure of specific SGID BWSPHs to 21-day differentiated Caco-2/HT-29MTX co-cultured cells, cell basolateral was subsequently observed to exhibit anti-adipogenic and adipolytic activities in 3T3-L1 cellular models. In addition, exposure of 3T3-L1 preadipocytes to hydrolysate BW-SPH-A during differentiation also increased GSH concentration upon stimulation with antioxidant tBOOH compared with the tBOOH control (p<0.05). Specific BWSPHs were also observed to reduced adiponectin production in LPS-stimulated cells compared with the LPS control (p<0.05). To conclude, certain BWSPHs exhibited significant bioactivities before SGID, after SGID, and after simulated intestinal absorption, therefore may have potential as health-enhancing functional food ingredients.
Fish , Protein hydrolysate , Bioactivity , Cell culture
Heffernan, S. 2022. Assessment of blue whiting protein hydrolysate bioactivities using cell culture models. PhD Thesis, University College Cork.