Biochemical fate of vicilin storage protein during fermentation and drying of cocoa beans

• pI-shift of the vicilin protein was observed during the onset of fermentation.
• Phophorylation and glycosylation observed in vicilin protein subunits.
• Cocoa proteins undergo proteolysis during fermentation with an initial increase and subsequent decrease in the diversity of peptides.
• Relative rate of degradation of different vicilin subunits varied across fermentation.

Key cocoa-specific aroma precursors are generated during the fermentation of cocoa beans via the proteolysis of the vicilin-like globulin. Previous studies had shown that degradation of this particular 566 amino acid-long storage protein leads to three distinct subunits with different molecular masses. Although oligopeptides generated from the proteolysis of vicilin-like globulin have been studied previously, changes occurring to vicilin at different stages of fermentation have not yet been explored in detail. The aim of this study was to investigate the fate of vicilin protein from the non-fermented stage up to the dried cocoa beans. Our results showed a remarkable shift in the electrophoretic mobility of vicilin towards higher pI during the onset of fermentation. The pI-shifted subunit was found susceptible to further degradation into a lower-molecular-weight vicilin subunit. The observed pI shift correlated with, but did not depend on protein phosphorylation. Glycosylation of some but not all vicilin subunits occurred at different stages of the fermentation process. Peptides generated from vicilin throughout fermentation were analyzed by UHPLC-ESI-MS/MS revealing an initial increase and subsequent decrease in the diversity of peptides with an increasing degree of fermentation. We furthermore describe the rate of degradation of different vicilin subunits. The detected diversity and dynamics of vicilin peptides will help to define biochemical markers of distinct steps of the fermentation process.

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