Biotinylated caffeic acid covalent binding with myofibrillar proteins in alkaline conditions: Identification of protein-phenol adducts and alterations in protein properties.

In this study, the effects of covalent interactions between myofibrillar proteins (MP) and caffeic acid (CA) were investigated. Protein-phenol adducts were identified by biotinylated caffeic acid (BioC) used as a substitution of CA. The total sulfhydryls and free amines content were decreased (p < 0.05). The α-helix structure of MP increased (p < 0.05) and MP gel properties enhanced slightly at low dosages of CA (10 and 50 µM), and both were impaired significantly (p < 0.05) at high dosages of CA (250 and 1250 µM). Two prominent adducts of myosin heavy chain (MHC)-BioC and Actin-BioC were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which gradually increased at low concentrations of BioC (10 and 50 µM), and raised significantly at the concentration of 1250 µM. According to the correlation analysis, MHC-BioC and Actin-BioC adducts showed a significant negative correlation with gel properties, such as G', hardness, and water holding capacity (WHC) (p < 0.01), which indicated that the covalent interactions between MP and CA significantly affected the quality of meat products.

Identification of Protein-Phenol Adducts in Meat Proteins: A Molecular Probe Technology Study.

Plant polyphenols with a catechol structure can form covalent adducts with meat proteins, which affects the quality and processing of meat products. However, there is a lack of fast and effective methods of characterizing these adducts and understanding their mechanisms. This study aimed to investigate the covalent interaction between myofibrillar protein (MP) and caffeic acid (CA), a plant polyphenol with a catechol structure, using molecular probe technology. The CA-MP adducts were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected via Western blot and LC-MS/MS analyses. The Western blot analysis revealed that various specific adducts were successfully enriched and identified as bands around 220 kDa, 45 kDa, and two distinct bands between 95 and 130 kDa. Combined with the LC-MS/MS analysis, a total of 51 peptides were identified to be CA-adducted, corresponding to 31 proteins. More than 80% of the adducted peptides carried one adducted site, and the rest carried two adducted sites. The adducted sites were located on cysteine (C/Cys), histidine (H/His), arginine (R/Arg), lysine (K/Lys), proline (P/Pro), and N-terminal (N-Term) residues. Results showed that the covalent interaction of CA and MP was highly selective for the R side chain of amino acids. Moreover, the adducts were more likely to form via C-N bonding than C-S bonding. This study provides new insights into the covalent interaction of plant polyphenols and meat proteins, which has important implications for the rational use of plant polyphenols in the meat processing industry.