HPLC and immunochemical detection of gliadin impurities in wheat and wheat products

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McCarthy, P.K., 1990. HPLC and immunochemical detection of gliadin impurities in wheat and wheat products. PhD, Nottingham Trent University.

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Abstract

Gliadin RP-HPLC of hexaploid wheat is shown to give "fingerprint type" profiles which are suitable for wheat variety identification. These profiles are found to reflect the genetic exchanges occuring during the crossing of wheat varieties in F1 hybrid wheat production. Two RP-HPLC techniques are proposed for PI hybrid purity determination; the first distinguishes genetically different seeds types from FI hybrid seed by their gliadin profiles and purity is presented as the percentage of FI hybrid seeds in a mixture; the second utilises the gliadin RP-HPLC profile of a batch of seeds by comparing the peak area ratio of unique gliadin peaks to that obtained using standard blends corresponding to 100-50% hybrid purity. The total analysis time (2nd method) is less than four hours and the results compare favourably with those obtained using acid PAGE of gliadins.

When hexaploid wheat gliadins are separated by RP- HPLC under identical conditions a major doublet peak elutes at 47.20+/-0.33 min and 47.94+/-0.05 min which is absent in the gliadin RP-HPLC profiles of pure Durum wheat. This is used as the basis of a method to detect the level of common wheat adulteration in Durum wheat and pasta products. It has been found that protein denaturation occurring during high temperature processing leads to some distortion of the early eluting peaks of the RP-HPLC profile, but the area of interest for detection of common wheat adulteration of pasta is unaffected. For Durum samples containing low levels of adulteration electrophoresis of fractions collected from RP- HPLC is proposed as a means of improving sensitivity. It is shown that with the possible exception of acid PAGE of gliadins, the current methods used for detecting common wheat adulteration of pasta products, are inadequate. This effect is clearly seen in the alkaline PAGE and IEF patterns of polyphenoloxidases and esterases respectively, where the loss of enzymatic activity and resolution make it difficult to detect the presence or absence of common wheat in pastas dried at high temperature.

The biochemical characteristics (electrophoretic mobilities, 2-D PAGE patterns, isoelectric points and molecular weights) of the γ/β gliadins eluting between 47-49 min on RP-HPLC separation of hexaploid wheat specific gliadin are presented. Polyclonal antisera raised to γ-gliadin 44 and 46 (eluting between 47-49 min) cross-reacts specifically with γ-gliadins found in hexaploid wheat and Durum pasta adulterated with common wheat. An immunochemical method is proposed using this antibody preparation to identify traces of common wheat adulteration in gliadin extracts of Durum wheat or pasta electrophoretically separated and immobilised on nitrocellulose paper.

Item Type:

Thesis

Creators:

McCarthy, P.K.

Date:

1990

ISBN:

9781369324280

Identifiers: