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Title: Studies on peptides from soya and meat proteins
Author: Crawford, F. J.
Awarding Body: University College of Swansea
Current Institution: Swansea University
Date of Award: 1983
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Several chapters in this thesis deal with the background to methods involved in identification of soya protein in meat products. The analytical method was introduced in 1978 by the laboratory of the Government Chemist in collaboration with Unilever Research laboratories for the detection of soya protein in cooked meat products. The analytical method involved digestion of the protein with trypsin, releasing a complex peptide mixture which was analysed on an ion-exchange column (Aminex A5). Two non-overlapping peaks SP-2 and MP-1 were characterised as being derived from the soya and meat component. The aim of the current project was to synthesise an internal standard for this analytical system. The fraction SP-2 and MP-1 were collected and found to contain minute amounts of peptide and a high concentration of citrate buffer (at about 5.6 pH) and additives such as BRIJ and n-octanoic acid. Chromatography revealed SP-2 and MF-1 to be composed of a mixture of peptides. Desalting (i.e the removal of citrate buffer) was the first priority. Partial success was achieved by passing the sample through gel columns (Sephadex 0-10 and Bio Gel P-2) which had fairly low molecular weight exclusion limits. Most of the salt could be removed in this way and it was found necessary to remove the BRIJ additive since it interfered in mass spectrometry studies. An improvement in the extractive methodology was achieved by subjecting the crude peptide contaminated with citrate buffer to a derivatisation sequence involving esterification with methanolic HC1 which converted the peptide, to the methyl ester hydrochloride and the citrate buffer to trimethylcitrate. The latter was extracted with chloroform. This left the peptide ester hydrochloride available for further derivatisation and mass spectroscopic analysis. Amino acid analysis was performed to determine which amino acids were present in the mixture of peptides existing. Acetylation/permethylation and trifluoroacetylation/esterification techniques were used to volatilise the peptides for their sequence determination. Direct insertion to the mass spectrometer, slow distillation of the probe and g.c-m.s were all used for the sequence determination of the mixture of peptides. These techniques were first studied using standard peptides. Techniques such as F.A.B, CI and EI mass spectroscopy were used to identify the mixture of peptides. Once the identification of a peptide from SP-2 mixture was made its synthesis was achieved using solid phase and later solution phase. Iuysine peptides such as Ala-Gly-Gly-I4ysOMe and Ala-Gly-Ser-I{ysCMe were found to be good standards on the Beckman analytical system. The peptides Ala-Gly-SerGlu(OMe)2 and Ala-Gly-Gly-LysOMe were found to be good internal standards on the Aminex A5 system (at the L.G.C). These could form the basis of internal standards for more accurate assessment of soya peptide content from tryptic digests of meat products. However further studies should be made to optimise the exact form of the peptides which would be suitable for application to h.p.l.c and similar techniques.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available