Characterisation and bioactive properties of protease inhibitors and phenolic components from Australian Wattle (ACACIA VICTORIAE BENTHAM) seed

Wattle seed (Acacia victoriae Bentham) is one of the most economically viable Australian native plants and has considerable potential as a food ingredient in raw and roasted forms. In this study, its minor constituents, namely protease inhibitors (PIs) and phenolic components were isolated and characterised. The effects of roasting on the chemical composition and their characteristics were also studied. Furthermore, extracts from raw and processed wattle seeds were analysed for in vitro bioactive properties.An Acacia victoriae trypsin inhibitor (AvTI) was purified from the seeds of prickly wattle (A. victoriae Bentham) by salt precipitation, ion exchange, and gel filtration chromatography and then characterised by electrophoresis, N-terminal amino acid sequencing, amino acid analysis and kinetic properties. AvTI had a specific activity of 138.99 trypsin inhibitor units per milligram (TIU mg-1), which was 21-fold higher than that of the salt precipitate. A molecular mass of 13 kDa with glycosylation of 2.06% was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, which also indicated that AvTI may consist of two polypeptide chains linked by at least one disulphide bond. Although only a single peak was resolved by ion exchange and reverse phase high-performance liquid chromatography (RP-HPLC), native-PAGE and isoelectric focusing revealed the presence of three isoforms possessing acidic pI values of 5.13, 4.76, and 4.27, respectively. AvTI had mainly glutamate, aspartate, leucine and lysine while the contents of sulphur-containing amino acids were low. N-terminal amino acid sequencing analysis of native and reduced AvTI showed two sequences with a high degree of homology with a typical Kunitz-type trypsin inhibitor.