Abstract
Rapeseed oil-extracted expeller cake mostly contains protein. Various approaches have been used to isolate, detect and measure proteins in rapeseeds, with a particular focus on seed storage proteins (SSPs). To maximize the protein yield and minimize hazardous chemical use, isolation costs and the loss of seed material, optimization of the extraction method is pivotal. For some studies, it is also necessary to minimize or avoid seed-to-seed cross-contamination for phenotyping and single-tissue type analysis to know the exact amount of any bioactive component in a single seed, rather than a mixture of multiple seeds. However, a simple and robust method for single rapeseed seed protein extraction (SRPE) is unavailable. To establish a strategy for optimizing SRPE for downstream gel-based protein analysis, yielding the highest amount of SSPs in the most economical and rapid way, a variety of different approaches were tested, including variations to the seed pulverization steps, changes to the compositions of solvents and reagents and adjustments to the protein recovery steps. Following SRPE, 1D-SDS-PAGE was used to assess the quality and amount of proteins extracted. A standardized SRPE procedure was developed and then tested for yield and reproducibility. The highest protein yield and quality were obtained using a ball grinder with stainless steel beads in Safe-Lock microcentrifuge tubes with methanol as the solvent, providing a highly efficient, economic and effective method. The usefulness of this SRPE was validated by applying the procedure to extract protein from different Brassica oilseeds and for screening an ethyl methane sulfonate (EMS) mutant population of Brassica rapa R-0-18. The outcomes provide useful methodology for identifying and characterizing the SSPs in the SRPE.
Highlights
Seeds of rapeseed species Brassica rapa, B. napus and B. juncea are utilized primarily for oil production
Following the establishment of a method, two biological replicates of single seeds were sampled from each of 2192 accession packages of the B. rapa genotype R-o-18 mutant Targeting Induced Local Lesions in Genomes (TILLING) population [26], obtained from the non-genetically modified M3 line of an M0 line that was chemically mutated with ethyl methane sulfonate (EMS), collected in the summer of 2010 at the University of Nottingham, Sutton Bonington campus, United Kingdom
The extracted proteins were separated by SDS-PAGE using either a Tris-Glycine or Tris-Tricine gel buffer system
Summary
Seeds of rapeseed species Brassica rapa, B. napus and B. juncea are utilized primarily for oil production. This globally important agronomic and bioeconomic crop is the second-largest cultivated oilseed around the world, supplying about 15% of the global consumption of edible vegetable oils [1,2]. The proteins constitute up to 50% of the seed and remain in the seed meal following oil extraction as a waste stream product [7]. The potential beneficial functional properties of these proteins, such as high solubility, water and fat binding, foaming, film forming and gelling abilities, make them sought after in the food processing industry [12,15,16]
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