Ruminant Milk Whey Proteome in Nutraceutical Research and Development
Ruminant milk whey, a by-product of cheese manufacture, is known to contain a large number of proteins. Bovine cheese whey at present is mostly used as nutritional supplements due to its high protein content, while non-bovine cheese whey such as that of sheep and goat has been mainly disposed of by land-spreading. However, research in the last few decades has led to an increasing recognition of whey proteins having functionalities with health benefits beyond nutritional values. Hydrolysis of whey proteins by the gastrointestinal tract following consumption is thought to generate a substantial number of bioactive peptides which can influence various physiological systems. As utilisation of cheese whey is potentially of significant benefit to the New Zealand economy, research in this study aimed to characterise the whey proteome of four ruminant species and assess the potential of whey proteins as a natural and inexpensive biomaterial in research and development of food products with health promoting benefits (also known as functional foods or nutraceuticals). Although the whey proteome of various ruminant species has been shown to comprise hundreds of proteins, a small number of those namely beta-lactoglobulin, alpha-lactalbumin, lactoferrin, serum albumin and immunoglobulins constitute approximately 90% of the total whey protein content. The major whey proteins of cow, sheep, goat and red deer milk were characterised and compared using one dimensional- and two dimensional-polyacrylamide gel electrophoresis (1D- and 2D-PAGE respectively), reversed phase- high performance liquid chromatography (RP-HPLC), ammonium sulfate fractionation and cation exchange chromatography. The whey proteins of the four species were demonstrated to differ in their physicochemical characteristics, indicating differences in amino acid composition. The whey proteins of red deer milk were found to be distinctly different from proteins of the other three species with regard to hydrophobicity and electrophoretic mobility characteristics displayed by RP-HPLC and 2D-PAGE respectively. The relative abundance of the major whey proteins was also found to vary between the four species. The abundance dynamic range of the whey proteome has been shown to present a technological challenge for identification of the whey proteins present in very low abundance. In this study, abundance normalisation of whey proteins of the four ruminant species was achieved using a ProteoMiner kit which utilises a combinatorial hexa-peptide ligand library technology. A combination of two electrophoretic fractionation methods namely 1D-PAGE and OFFGEL isoelectric focusing and LCMS/MS of the ProteoMiner treated whey samples resulted in identification of a substantial number of proteins in whey of each of the four species (553 for cow, 606 for sheep, 1133 for goat and 491 for red deer). Gene ontology analysis of the whey proteins revealed that while the proteins common to whey of the four species were significantly enriched for biological processes involving immunity and inflammatory responses, whey proteins unique to each of the four species were enriched for cellular establishment, metabolic processes and regulatory functions. The identification of a substantial number of whey proteins of the four species also enabled a more thorough analysis of the evolution of the ruminant lactating proteome. Using an established phylogenetic tree of the ruminantia, the four ruminant species were found to recruit and dismiss different sets of proteins to and from their milk whey proteome along the course of evolution. The proteins common to whey of the four ruminant species were also subjected to sequence homology analysis using the human proteome as reference in order to investigate the rate of evolution of the ruminant lactating proteome. Due to a lack of a red deer genomic sequence, the sequence homology analysis was limited to proteins of cow, sheep and goat whey. Although the overall sequence homology of whey proteins was found to be similar in the three species, the sequence homology percentile of each individual whey protein compared to the human analogue appeared to vary across the three species, suggesting that individual whey proteins of each of the three species evolved at different rates. A combination of functional classification and sequence homology analyses indicated that the rate of protein evolution was associated with their function in milk. These analyses also showed that across the three species, the protein components in each of the functional groups shared a similar overall sequence homology percentile when compared to their human analogues, suggesting that the function-evolutionary rate relationship was independent of species. Bovine whey proteins were assessed for their potential in development of functional foods using various food-grade protease preparations. Two fungal proteases were found to be able to extensively hydrolyse bovine whey proteins while leaving the most abundant and most reported allergenic whey protein beta-lactoglobulin intact. The protease preparations were found to contain a mixture of proteases using 1D-PAGE, 2D-PAGE and protein identification with mass spectrometry. The hydrolytic specificity of the protease preparations were characterised using BODIPY-FL casein as substrate. As intact beta-lactoglobulin has been shown to possess characteristics suitable for various applications in industry, the integrity of beta-lactoglobulin isolated from whey protein hydrolysates using membrane ultrafiltration was examined using 1D- and 2D-PAGE, RP-HPLC and protein intact mass spectrometry. Bovine whey peptide hydrolysates, following removal of beta-lactoglobulin, were subjected to bioactivity screening using antioxidant, angiotensin converting enzyme inhibition, cytokine, lymphocyte proliferation and antibacterial assays. The peptide hydrolysates were found to exhibit significant bioactivities in comparison with the unhydrolysed whey sample. Sequential sub-fractionation of the peptide hydrolysates using gel permeation chromatography and OFFGEL isoelectric focusing resulted in multiple fractions with contrasting activities, demonstrating the diversity of the bioactive peptides present in the hydrolysates. Bioactivity screening of the fractions generated also suggested a synergistic mechanism of action of the bioactive peptides. This project using technological tools from multiple disciplines examined and compared the diversity of the milk whey proteome of various ruminant species, and demonstrated the potential of whey proteins in the research and development of nutraceutical products.
Advisor: Carne, Alan; Bekhit, Alaa El-Din; McConnell, Michelle
Degree Name: Doctor of Philosophy
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: Milk; Proteome; Bioactivity; Deer; Whey
Research Type: Thesis