Generation and characterisation of biologically active milk-derived protein and peptide fractions

In recent years, extensive research has been carried out on the health benefits of milk proteins and peptides. Biologically active peptides are defined as specific protein fragments which have a positive impact on the physiological functions of the body; such peptides are produced naturally in vivo, but can also be generated by physical and/or chemical processes, enzymatic hydrolysis and/or microbial fermentation. The aims of this thesis were to investigate not only the traditional methods used for the generation of bioactive peptides, but also novel processes such as heat treatment, and the role of indigenous milk proteases, e.g., in mastitic milk, in the production of such peptides. In addition, colostrum was characterised as a source of bioactive proteins and peptides. Firstly, a comprehensive study was carried out on the composition and physical properties of colostrum throughout the early-lactation period. Marked differences in the physico-chemical properties of colostrum compared with milk were observed. Various fractions of colostrum were also tested for their effect on the secretion of pro- and anti-inflammatory cytokines from a macrophage cell line and bone marrow dendritic cells, as well as insulin secretion from a pancreatic beta cell line. A significant reduction in the secretion of the pro-inflammatory cytokines, TNF-α, IL-6, IL-1β and IL-12, a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, as well as a significant increase in insulin secretion were observed for various colostrum fractions. Another study examined the early proteomic changes in the milk of 8 cows in response to infusion with the endotoxin lipopolysaccharide (LPS) at quarter level in a model mastitic system; marked differences in the protein and peptide profile of milk from LPS challenged cows were observed, and a pH 4.6-soluble fraction of this milk was found to cause a substantial induction in the secretion of IL-10 from a murine macrophage cell line. Heat-induced hydrolysis of sodium caseinate was investigated from the dual viewpoints of protein breakdown and peptide formation, and, a peptide fraction produced in this manner was found to cause a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, from a murine macrophage cell line. The effects of sodium caseinate hydrolysed by chymosin on the gut-derived satiety hormone glucagon-like peptide-1 (GLP-1) were investigated; the resulting casein-derived peptides displayed good in vitro and in vivo secretion of GLP-1. Overall, the studies described in this thesis expand on current knowledge and provide good evidence for the use of novel methods for the isolation, generation and characterisation of bioactive proteins and/or peptides.

Nutraceutical and functional food bioactive peptides in beef

In recent years, the potential to positively modulate human health through dietary approaches has received considerable attention. Bioactive peptides which are released during the hydrolysis or fermentation of food proteins or following digestion may exert beneficial physiological effects in vivo. The aim of this work was to isolate, characterise and evaluate Angiotensin-І-converting enzyme (ACE-І) inhibitory, antimicrobial and antioxidant peptides from the bovine myofibrillar proteins actin and myosin. In order to generate these peptides, the myofibrillar proteins actin and myosin were hydrolysed with digestive enzymes pepsin, trypsin and α-chymotrypsin, or with the industrial thermolysin-like enzyme “Thermoase”, Amano Inc. It was found that each hydrolysate generated contained peptides which possessed ACE inhibitory, antioxidant and antimicrobial activity. The peptides responsible in part for the observed ACE inhibitory, antioxidant and antimicrobial activity of a number of hydrolysates were isolated using the method of RP-HPLC and the bioactive peptides contained within each active fraction was determined using either MALDI-TOF MS/MS or N-terminal peptide sequencing. During the course of this thesis six ACE inhibitory and five antimicrobial peptides were identified. It was determined that the reported antioxidant activity was a direct result of a number of peptides working in synergy with each other. The IC50 values of the six ACE inhibitory peptides ranged in values of 6.85 to 75.7 µM which compare favourably to values previously reported for other food derived ACE inhibitory peptides, particularly the well known milk peptides IPP and VPP, IC50 values of 5 and 9 µM respectively. All five antimicrobial peptides identified in this thesis displayed activity against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Listeria innocua with MIC values ranging from 0.625 to10 mM. The activity of each antimicrobial peptide was strain specific. Furthermore the role and importance of charged amino acids to the activity of antimicrobial peptides was also determined. Generally the removal of charged amino acids from the sequence of antimicrobial peptides resulted in a loss of antimicrobial activity. In conclusion, this thesis revealed that a range of bioactive peptides exhibiting ACE inhibitory, antioxidant and antimicrobial activities were encrypted in bovine myofibrillar proteins that could be released using digestive and industrial enzymes. Finally enzymatic hydrolysates of muscle proteins could potentially be incorporated into functional foods; however, the potential health benefits would need to be proven in human clinical studies.