Abstract
There is increasing interest in the diversification of the traditional cattle-based dairy industry with milk and milk products from alternative lactating species, such as sheep, goat and red deer. Interest in the development of dairying with small ruminants and alternative lactating animals has arisen in part due to the apparent differences in the composition and physicochemical properties of milks from different species. Milk from alternative lactating animals is mainly used for the production of special cheese, producing a whey co-product fraction which is known to contain a number of proteins that are being increasingly recognised for their nutritional value and potential as food ingredients.
Lactoferrin, lactoperoxidase and osteopontin are milk whey proteins of recognised health-promoting value as individual molecules, and there is increasing evidence for the formation of an osteopontin-lactoferrin-lactoperoxidase complex in milk. The relatively low abundance of these three proteins in milk (~10 mg/L) presents technical challenges for their identification and characterisation. Lactoperoxidase shares some similar molecular characteristics and properties with lactoferrin, including molecular weight and isoelectric point, which complicates the separation and enrichment of these proteins. Further, osteopontin is glycosylated and substantially phosphorylated, and is reported to be partially hydrolysed in the mammary gland by proteases naturally present in milk which, along with likely amino acid sequence differences across different species, also makes this protein more challenging to identify and analyse. As a result, there is limited information in the literature for lactoferrin, lactoperoxidase and osteopontin, especially from the milk of alternative non-cattle dairy species.
This study reports chromatography strategies that were developed for the selective enrichment of lactoferrin, lactoperoxidase and osteopontin from the milk of selected dairy species. Analysis by 1D SDS-PAGE of chromatography fractions revealed four candidate protein species migrating at approximately 80, 78, 55 and 40 kDa which were consistent with previous reports for the molecular weights of the cow milk whey proteins lactoferrin, lactoperoxidase, osteopontin and a truncated osteopontin fragment, respectively. The milk whey proteins of interest were found to vary in their relative abundance, but not apparent physicochemical properties, between several different dairy species. In the present study, liquid chromatography-coupled tandem mass spectrometry analysis has confirmed the putative candidates for the cow milk whey proteins lactoferrin, lactoperoxidase, osteopontin and the fragment of osteopontin. Proteomic analysis showed that the ~40 kDa osteopontin fragment is a truncated phosphorylated fragment of the full-length mature osteopontin protein, and is likely the result of proteolysis in the mammary gland.
Moreover, the highly enriched protein fractions were used in size-exclusion chromatography experiments to conduct a preliminary investigation of the molecular interactions that lactoferrin, lactoperoxidase and osteopontin are reported to form in milk. A complex of lactoferrin and osteopontin with a predicted 3:1 stoichiometry was demonstrated, for which the molecular weight and Stokes radius were determined to be different than that of the individual constituent proteins of the complex. Lactoperoxidase and osteopontin were not shown to interact under the experimental conditions of the study. The findings of the present study suggest that the N-terminal region of osteopontin, which the truncated osteopontin fragment is believed to be composed of, may be important for the formation of electrostatic interactions with lactoferrin in milk. However, the molecular characteristics of the proposed lactoferrin-lactoperoxidase-osteopontin complex and its biological significance still require further investigation.