Trypsin inhibitor content of New Zealand grown pea cultivars : A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science at Lincoln University, Canterbury, New Zealand

Abstract

The purpose of this study was to determine various components of field pea cultivars grown in Canterbury , New Zealand. Proximate analysis was used to measure the nutritional composition of these pea variaties. Of particular interest were the antinutritional components, trypsin inhibitors Comparison of different techniques to extract and quantify trypsin inhibitors, and purification of various trypsin inhibitor isoforms were the major focus of this study. The effect of location variation was also investigated to determine if environmental conditions significantly altered the composition of the pea cultivars. The proximate composition (protein, fibre, fat and ash content) of thirteen pea cultivars resulted in significant differences (p<0.05) between all cultivars. In addition the effect of growing four pea cultivars at three different locations exerted a significant (p<0.05) effect on the protein, fibre and ash content of all cultivars studied. Measurement of trypsin inhibitor content (using the Kakade et al,(1974) enzymic assay), of pea cultivars grown at Lincoln varied significantly (p<0.05). The majority of cultivars contained less than 2 mgTI/g pea flour, which is the maximum level soon be allowed for export to the European Union. The range of trypsin inhibitor content of these New Zealand grown peas was 0.32 - 2.57 mgTI/g pea flour. Location also significantly (p<0.05) altered the trypsin inhibitor content of the four pea cultivars grown at Chertsey and Methven. The amount of trypsin inhibitor in peas was also determined using ELISA. The results from ELISA correlated well (R²=0.82) with the traditional Kakade (et al, 1974) enzymic assay. Five proteinase inhibitors were purified from the pea extracts firstly by gel filtration and then by ion exchange chromatography. Isolation of two peaks IV and V had mobility on SDS-PAGE equivalent to 29-30 kDa. As other researchers have suggested trypsin inhibitors can bind together, it is possible the trypsin inhibitor molecules in the isolated fractions may be associated in multiple units. Isoelectric focusing of eight pea cultivars identified between three and nine isoforms of trypsin inhibitor. Generally, cultivars that contained high levels of trypsin inhibitor also contained a large number of isoinhibitors. A maximum of nine trypsin isoinhibitors were detected in two cultivars (Birte and Prussian Blue) grown at Chertsey, however only three isoinhibitors were detected in the two maple pea varieties measured. The range of isoelectric points obtained from the inhibitors of pea extracts were from pI 4. 7-9 .4.