Origin and varietal based proteomic and peptidomic fingerprinting of Theobroma cacao in non-fermented and fermented cocoa beans.

It is well known that the development of chocolate flavor is initiated during cocoa bean fermentation. Storage proteins undergo the most intensive breakdown yielding peptides and free amino acids, which both serve as flavor precursors. A comprehensive analysis of cocoa proteins and oligopeptides of non-fermented and fermented beans from various geographic origins allows the assessment of systematic differences with respect to their origin as well as fermentation status. Protein quantities as well as their profiles derived from two-dimensional gel electrophoresis, showed striking differences for non-fermented beans depending on their geographical origin. From fermented beans, oligopeptides were relatively quantified by utilizing UHPLC-ESI-Q-q-TOF and annotated based on their characteristic fragmentation pattern in the positive-ion mode. With >800 unique oligopeptides, excluding di- and tri-peptides, across 25 different samples, we are herein reporting on the largest collection of cocoa oligopeptides ever observed and identified. The detected diversity of peptides could not be correlated to the geographical origin but rather to the degree of fermentation. Our findings suggest that the variability in peptide patterns depends on the fermentation method applied in the country of origin ultimately indicating diversified proteolytic activities. Furthermore, our results showed that well-fermented and fair-fermented beans can be differentiated from partially fermented and under-fermented ones by higher numbers and total amounts of oligopeptides.

Comparative validation of a microcapsule-based immunoassay for the detection of proteins and nucleic acids.

To detect and study diseases, research and clinical laboratories must quantify specific biomarkers in the plasma and urine of patients with precision, sensitivity, and cost-effectiveness. Newly developed techniques, such as particle-based immunoassays, must be validated in these terms against standard methods such as enzyme-linked immunosorbent assays (ELISAs). Here, we compare the performance of assays that use hollow polyelectrolyte microcapsules with assays based on solid plastic beads, and with standard microplate immunoassays. The polyelectrolyte microcapsules detect the disease biomarker beta-2 microglobulin with a fifty-fold increase in sensitivity than polystyrene (PS) beads. For sequence-specific nucleic acid detection, the oligonucleotide-coated microcapsules exhibit a two-fold lower increase in sensitivity over PS beads. The microcapsules also detect the presence of a monoclonal antibody in hybridoma supernatant at a fifty-six-fold increase in sensitivity compared to a microplate assay. Overall, polyelectrolyte microcapsule-based assays are more sensitive for the detection of protein and nucleic acid analytes than PS beads and microplate assays, and they are viable alternatives as a platform for the rapid quantitative detection of analytes at very low concentrations.