Development Of Proteomic Methodologies For Malting Barley Cultivar Discrimination

The properties and cultivars of malting barley are closely related with malting and brewing process. The qualities of barley are the key determinant factors of the final beer product. A fast, sensitive and quantitative method of barley cultivar discrimination is in great needed for maltsters. Proteomics and quantitative real-time PCR(q RT-PCR) were introduced for malting barley cultivar discrimination and purity identification in the present work.Hordein, as protein marker of barley cultivars, was sequential extracted in the light of solubility properties of different protein components in barley seeds. In the present study, seven commonly used Australia malting barley cultivars(Gairdner, Baudin, Hindmarsh, Vlamingh, FAQ, Schooner, and Commander), and four Chinese barley(Supi#3 and Supi#6, Kenpi#7 and Kenpi#10) in Chinese brewing industry were selected for the new barley cultivar discrimination method development. The hordeins two-dimensional electrophoresis(2DE) profile of each barley cultivar was obtained, and constituted the hordein standard profiles library of the eleven barley cultivars. Distribution patterns and number of protein spots among the seven Australia barley cultivars showed obvious differences, as well as the Australia and Chinese barley cultivars. The differences could be utilized to discriminate different barley cultivars.By comparing the hordein 2DE profiles using PDQuest, 43 common protein spots and 18 specific protein spots that presented in only one certain barley cultivar gel were found, and 34 common protein spots and 13 specific protein spots were successfully identified by matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF/TOF), which comprised of the protein markers library of the seven Australia barley. Common protein spots were mostly identified as B, C and D group hordein, or hordein precursors. A large number of α-amylase inhibitor and trypsin inhibitor family, also called chloroform/methanol-soluble protein, and some other unnamed and hypothetical proteins were also identified. Specific protein spots presented only in the 2DE profile of one certain barley cultivar were considered to be the representative hordein markers of this variety were identified as follows: Gairdner for Hordein B, α-amylase/trypsin inhibitor CM 16 and α-amylase inhibitor BMAI-1, Baudin for B3-hordein and B1-hordein, Vlamingh for α-amylase/trypsin inhibitor CMa, hordoindoline A-1 and CMd preprotein(AA-14 to 146), Schooner for BTI-CMe 2.2 protein and α-amylase inhibitor BDAI-1.The standard curves regarding expression level of hordein markers to purity of barley cultivar Gairdner were set by artificial hybrid tests with q RT-PCR. Finally, the q RT-PCR method was compared with the conventional SDS-PAGE method by double-blind experiment, and the former one was proved to be as accurate as the conventional method.The results indicated that the new developed method for barley cultivar identification with 2DE profiles of hordeins and purity calculation with q RT-PCR of the representative hordein marker was effective and applicable in malting and brewing.