Antimicrobial peptides isolated from ovine blood neutrophils : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand

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The aim of the research presented in this thesis was to investigate the properties of the antimicrobial peptides found in ovine blood, in order to assess their potential as a high-value product. Due to the large number of lambs and sheep that are slaughtered New Zealand (approximately 25 million lamb and 5 million sheep per year), there are considerable volumes of ovine blood available for processing (approximately 40 million litres per year). Currently this blood is dried and sold as a low value product. The first objective of this research was to purify and characterise the antimicrobial peptides isolated from ovine neutrophils. A number of proline/arginine-rich peptides, as well as two small fragments of larger proteins, that displayed antimicrobial activity were identified. The second objective of this research was to investigate the mechanism of action of ovine antimicrobial peptides. For this investigation, three ovine peptides, α-helical SMAP29 and proline/arginine-rich OaBac5mini and OaBac7.5mini, were synthesised. Of these, SMAP29 was the most potent. The three peptides all bound Gram-negative bacterial LPS and caused the outer membrane to be permeabilised. SMAP29 caused significant depolarisation of the cytoplasmic membrane that led to cell lysis. However, the other two peptides only caused slight depolarisation of the cytoplasmic membrane, which indicates that they probably passed through the membrane to interact with the inner cellular contents. The third objective of this research was to investigate the morphological changes to bacterial cells induced by the ovine antimicrobial peptides. Transmission electron microscopy and atomic force microscopy confirmed that SMAP29 caused significant damage to the membranes of bacterial cells and induced cell lysis; whereas, OaBac5mini caused minor alterations to the bacterial membranes but did not induce cell lysis. The fourth objective of this research was to determine the effect of the environmental conditions on the activity of the peptides. The peptides were very stable over a range of pH values and when heated to temperatures up to 80°C. The activity of the peptides decreased slightly in the presence of monovalent cations and was inhibited by the presence of divalent cations. The peptides were significantly more active in combination than individually, and they were strongly synergistic with polymyxin B, a peptide antibiotic. The final objective of this research was to develop a pilot-scale extraction process for the isolation of antimicrobial peptides from ovine blood. The laboratory-scale process was simplified and adapted to design a process that could be used industrially. The crude pilot-plant extract was active against a broad-range of food pathogens and disease causing organisms. The antimicrobial peptides found in ovine blood have the potential to be used as biopreservatives for chilled lamb products, or in a topical cream for cuts and grazes; therefore it is recommended that further research is carried out to investigate the above applications and. if successful, the feasibility of commercialising the technology.
Content removed due to copyright restrictions: Anderson, R.C., Wilkinson, B. & Yu, P.L. (2004). Ovine antimicrobial peptides: new products from an age-old industry. Australian Journal of Agricultural Research, 55(1),69-75. Anderson, R.C. & Yu, P.L. (2003). Isolation characterisation of proline/arginine-rich cathelicidin peptides form ovine neutrophils. biochemical and biophysical research communications 312(4), 1139-1146. Anderson, R. C., Hancock, R.E.W. & Yu, P.L. (2004). Antimicrobial activity and bacterial membrane interaction of ovine-derived cathelicidins. Antimicrobial Agents and Chemotherapy, 48(2), 673-676. Anderson, R.C., Haverkamp R. & Yu, P.L. (2004). Investigation of morphological changes to S. aureus induced by ovine-derived antimicrobial peptides using TEM and AFM. FEMS Microbiology Letters, 240(1), 105-110. Anderson, R.C. & Yu, P.L. (2005). Factors affecting the antimicrobial activity of ovine-derived cathelicidins against E. coli 0157:H7. International Journal of Antimicrobial Agents 25, 205-210
Ovine peptides, Peptide extraction, Ovine blood use