Improving solubility and functional properties of phycocyanin under acidic conditions by glutaminase deamidation and succinylation

The use of phycocyanin (PC), a protein-based blue colorant, in acidic food products is limited by its susceptibility to aggregation at, and around, its isoelectric point (pI similar to 4). We modified the pI and functional properties of PC by succinylation and protein-glutaminase (PG) deamidation. The conditions for deamidation and succinylation were systematically optimized based on attaining a high degree of modification while maintaining low PC color loss and hydrolysis during reactions. A new protocol combining acidification with mild heat treatment was developed to minimize PC denaturation while ensuring efficient termination of PG deamidation. Under optimal conditions, succinylation and deamidation enhanced the electronegativity of PC at pH 4-7. Circular dichroism and electrophoresis revealed the unfolding of PC secondary structure and the disassembly of large aggregates into small fragments after modification. Succinylation further isolated the coiled PC helices into single strands. These structural extensions improve the molecular flexibility and exposed the buried hydrophobic groups, particularly in succinylated PC. After deamidation and succinylation, PC showed increased solubility, surface hydrophobicity, emulsifying and foaming abilities, and maintained good color stability upon heating at pH 4 and 5; these gains were higher for succinylated PC. Unfortunately, at pH 3, misfolding and massive aggregation occurred in both modified samples, making their solubility and heat stability at pH 3 noticeably worse than unmodified PC. However, the improved solubility, heat stability, and functional properties between pH 4 and 5 will allow the modified PC to survive acidic environments and heat processing conditions for many common foods.