Epsilon Archive for Student Projects

Abstract

Red clover (Trifolium pratense L.) is an important forage crop with a high protein content and potential for further improvement through plant breeding. Reliable and reproducible methods for determining protein extractability are required to enable comparisons between different red clover accessions. The aim of this study was to develop and evaluate a method for determining protein extractability from freeze-dried red clover leaves.
Different extraction solutions and extraction conditions were evaluated, including water, sodium chloride, sodium hydroxide, phosphate buffer with and without sodium dodecyl sulfate, different extraction times and temperatures, and sonication. Soluble protein concentration in the extracts was quantified using bicinchoninic acid assay and selected extracts were qualitatively analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The developed method was subsequently applied to different red clover accessions.
Protein extractability varied between the extraction solutions, with 0,1 M NaCl resulting in the highest protein yield among the solutions evaluated. In comparison, extraction temperature, extraction time and sonication had less pronounced effects, and no consistent relationship between these parameters and protein extractability was observed under the conditions investigated. The method was also applied to different red clover accessions and revealed variation in extractable protein between them. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the presence of proteins in the analysed extracts and provided complementary qualitative information on their protein profiles.
Overall, the results demonstrate that the choice of extraction solution was an important factor affecting protein extractability from freeze-dried red clover leaves. The developed method shows potential for comparative analyses of protein extractability among red clover accessions. However, further validation using additional biological replicates and a larger number of accessions is required before the method can be applied in more extensive plant breeding studies.