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Characterization of oat proteins and aggregates using asymmetric flow field-flow fractionation

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Abstract

The soluble proteins and protein aggregates in Belinda oats were characterized using asymmetric flow field-flow fractionation (AF4) coupled with online UV–vis spectroscopy and multiangle light-scattering detection (MALS). Fractions from the AF4 separation were collected and further characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The AF4 fractogram of the oat extracts revealed three peaks which were determined to be monomeric forms of soluble proteins, globulin aggregates, and β-glucan, respectively. The early eluting monomeric proteins ranged in molar mass (MM) between 5 and 90 kg/mol and in hydrodynamic diameter (D h) from 1.6 to 13 nm. The MM at peak maximum of the globulin aggregate peak was found to be ∼300 kg/mol and the D h was measured to be ∼20 nm. SDS-PAGE of the collected fraction across this peak revealed two bands with MM of 37 and 27 kg/mol which correspond to the α and β subunits of globulin indicating the elution of globulin aggregates. A third peak at long retention time was determined to be β-glucan through treatment of the oat extract with β-glucanase and by injection of β-glucan standards. The amount of soluble protein was measured to be 83.1 ± 2.3 wt.%, and the amount of albumin proteins was measured to be 17.6 ± 5.7 wt.% of the total protein in the oats. The results for Belinda oat extracts show that the AF4-MALS/UV platform is capable of characterizing the physicochemical properties such as MM and hydrodynamic size distribution of proteins and protein aggregates within a complicated food matrix environment and without the need to generate protein isolates.

MALS (red) and UV (blue) asymmetrical flow field-flow fractionation (AF4) fractograms of Belinda oat extract (BOE) superimposed with SDS-PAGE analysis of collected fractions. The SDS-PAGE of unfractioned BOE is seen in the left most lane and the MM ladder is shown on the right. Separation of monomeric proteins from protein aggregates in the BOE is observed

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Acknowledgments

The authors would like to thank The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS, grant 222-2008-761) for project funding and Bindu A. Sunilkumar for guidance involving the silver staining of the SDS-PAGE gels.

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Authors and Affiliations

  1. Lund Centre for Field-Flow Fractionation, Department of Food Technology, Engineering and Nutrition, Lund University, P.O. Box 124, 221 00, Lund, Sweden

    J. Ray Runyon, Lars Nilsson, Johan Alftrén & Björn Bergenståhl

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  1. J. Ray Runyon
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  2. Lars Nilsson
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  3. Johan Alftrén
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  4. Björn Bergenståhl
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Correspondence to J. Ray Runyon.

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Runyon, J.R., Nilsson, L., Alftrén, J. et al. Characterization of oat proteins and aggregates using asymmetric flow field-flow fractionation. Anal Bioanal Chem 405, 6649–6655 (2013). https://doi.org/10.1007/s00216-013-7115-7

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  • DOI: https://doi.org/10.1007/s00216-013-7115-7

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