Abstract
In this study, the interaction of native and transglutaminase (Tgase) cross-linked β-lactoglobulin (β-LG) with caffeic acid (CA) was examined, aiming to obtain functional composites. Knowledge on the binding affinity and interaction mechanism was provided by performing fluorescence spectroscopy measurements, after heating the native and cross-linked protein at temperatures ranging from 25 to 95 °C. Regardless of the protein aggregation state, a static quenching mechanism of intrinsic fluorescence of β-LG by CA was established. The decrease of the Stern–Volmer constants with the temperature increase indicating the facile dissociation of the weakly bound complexes. The thermodynamic analysis suggested the existence of multiple contact types, such as Van der Waals’ force and hydrogen bonds, between β-LG and CA. Further molecular docking tests indicated the existence of various CA binding sites on the β-LG surface heat-treated at different temperatures. Anyway, regardless of the simulated temperature, the CA-β-LG assemblies appeared to be unstable. Compared to native protein, the CA-β-LG and CA-β-LGTgase complexes (ratio 1:1) exhibited significantly higher antioxidant activity and inhibitory effects on α-glucosidase, α-amylase, and pancreatic lipase, enzymes associated with metabolic syndrome. These findings might help the knowledge-based development of novel food ingredients with valuable biological properties.
Highlights
Caffeic acid (CA) is a polyphenol produced through the secondary metabolism of vegetables [1], including olives, coffee beans, fruits, potatoes, carrots, and propolis, and constitutes the main hydroxycinnamic acid found in the diet of humans
To the best of our knowledge, this is the first time that the complexation of β-LG, in different molecular states with CA is studied, aiming to obtain functional composites with enhanced antioxidant, inhibitory effects towards enzymes associated with metabolic syndrome, including α-glucosidase and α-amylase, and pancreatic lipase
The antioxidant activity of the CA-β-LG and CA-β-LGTgase samples was compared with the antioxidant activity of the native protein before complexation. β-LG showed antioxidant activity of 0.52 ± 0.06 mMol Trolox/g DW, whereas the 1:1 complexation led to a significant increase of up to 15.02 ± 0.40 mMol Trolox/g DW for CA-β-LG and 23.86 ± 0.76 mMol Trolox/g DW for CA-β-LGTgase
Summary
Caffeic acid (CA) is a polyphenol produced through the secondary metabolism of vegetables [1], including olives, coffee beans, fruits, potatoes, carrots, and propolis, and constitutes the main hydroxycinnamic acid found in the diet of humans. In this study, the binding mechanism between CA and β-LG in its native and Tgase-assisted cross-linked state (n-β-LG and Tgase-β-LG) was detailed by fluorescence spectroscopy and molecular modeling for the proper understanding of the functional biologically active compound-protein interactions, and, for estimating the potential therapeutic and technological applications. We hypothesized that the complexation of CA by β-LG in two molecular states, native and cross-linked, would lead to several complexes, in which the β-LG-CA complexes might present superior biological function, such as antioxidant and inhibitory effects on some enzymes involved in metabolic syndrome. To the best of our knowledge, this is the first time that the complexation of β-LG, in different molecular states with CA is studied, aiming to obtain functional composites with enhanced antioxidant, inhibitory effects towards enzymes associated with metabolic syndrome, including α-glucosidase and α-amylase, and pancreatic lipase
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