Rapid wheat varietal identification using the Agilent 2100 bioanalyzer and automatedpattern-matching

Dr Dhan BhandariApplication Note

Abstract

Accurate identification of wheat varieties is of paramount importance

to the milling industry in many countries. This Application Note

describes how the Agilent 2100 bioanalyzer and the Protein 230 assay

can be used in conjunction with a third-party software, to analyze

wheat proteins for varietal identification.

Agilent Equipment:

• 2100 Bioanalyzer Protein 230 Kit

Application Area:

• Food analysis

Figure 1Typical wheat protein separation by the Protein 230 assay.

varieties were analyzed. The elec-tropheromram profiles wereprocessed using the Phoretix 1DAdvanced and 1D Database (Non-linear Dynamics) software for pat-tern-matching purposes.

Introduction

Authentication of varieties isimportant for the cereal industryfor maintenance and testing ofgrain quality to meet marketrequirements. The charge-basedseparation of proteins by the acid-PAGE (polyacrylamide gel elec-trophoresis) technique is widelyused for wheat varietal identifica-tion. However, this requires highlyskilled operators to prepare, runand scan the gels and interpretband patterns. Also, there aresafety concerns regarding the tox-icity of unpolymerised acry-lamide. While acid-PAGE is effec-tive in analytical laboratories, theroutine method can take up totwo days. This is too slow for useat mill intake, which requiresassessment of the wheat shipmentduring the period of delivery –typically under one hour. In thisApplication Note we demonstratethe use of the Agilent 2100 assay,with bioanalyzer and Protein 230the Nonlinear Dynamics’ Phoretix1D Advanced (TotalLab TL120DM) computerized pattern-recog-nition software to provide a bet-ter alternative to acid-PAGE. Theaim of this study was to develop arobust, automated method forrapid identification of wheat vari-eties.

MethodsTotal wheat proteins (includingglutenins) were extracted fromindividual grains in 0.4 mL of 2Murea, 15 % glycerol, 0.1 M DTTand 0.1 M Tris/HCl, pH 8.8, usingan ultra-sonic water bath for 15minutes. Extracts were centri-fuged at 11,000 g for 5 minutesand treated with the Protein 230assay reagents in according to the

assay protocol. The samples wereseparated on the Agilent 2100 bio-analyzer, with each analysis of 10samples plus ladder taking lessthan 25 minutes. Replicates of 34wheat cultivars representing the2004/5 UK Recommended List

Lower Marker

HMW-Glutenins

α-, β-, γ- Gliadins

ω-Gliadins,L MW-Glutenins

Upper Marker

Figure 2Bioanalyzer gel-like image of MW standards and 10 different wheat varieties.

2

ResultsThe Protein 230 assay producedwell-resolved protein profiles, suit-able for varietal discrimination(figures 1 & 2). The Phoretix soft-ware was able to compare theelectropherogram profiles. Figure3 shows an example of a dendro-gram where all the replicates ofthree different varieties are cor-rectly grouped. A prototype wheatlibrary was developed by selectingthe most representative varietalprofile. Results showed that 90 %of test samples could be identifiedwithin the top three matches.Work is in progress to optimize theperformance of this library. Thepracticality of the method and therobustness of the system is bourneout by the fact that the system isnow in routine use in UK commer-cial mill intake laboratories.

ConclusionsOur study has demonstrated thatusing the Agilent 2100 bioanalyzerwith the Phoretix system offers astandardized, objective methodfor rapid varietal discrimination.The ease of use and total analysistime of less than 50 minutesmakes it most suitable for millintake use. The optimized systemwill enable millers to make moreconfident decisions in acceptinggrain consignments, and couldbecome widely adopted as aneffective policing tool within thegrain industry. A number of UKmills have purchased the com-bined systems for screeningwheat deliveries at intake.

Figure 3Dendrogram illustrating pattern-matching of replicate profiles of 3 wheat varieties.

3

AcknowledgementsCCFRA is grateful to the NationalAssociation of British and IrishMillers (nabim) for sponsoringthis study.

Dr Dhan Bhandari is Senior

Scientist at Campden & Chorley-

wood Food Research Association

(CCFRA), Chipping Campden,

Gloucestershire, GL55 6LD, UK.

www.agilent.com/chem/2100

© 2008, 2016 Agilent Technologies Inc.

Published March 1, 2016 Publication Number 5989-7735EN