Characterization of tea polyphenol-modified lactoferrin complexes and their antimicrobial activity against Escherichia coli

Lactoferrin is a bioactive dairy protein with multiple beneficial effects. The use of lactoferrin-based combined strategies is not only effective in combating pathogenic bacteria but also remarkably reduces the emergence of bacterial resistance, which is of great practical significance. In this study, gallic acid and epigallocatechin gallate, tea polyphenols with excellent antimicrobial effects, were selected to bind with lactoferrin (LF) in a non-covalent interaction mode. The complex was characterized using a series of spectral experiments and molecular docking, and its antibacterial activity was evaluated using Escherichia coli as a model. Ultraviolet, fluorescence, and infrared spectroscopy showed that gallic acid and epigallocatechin gallate mainly bind to lactoferrin hydrogen bonding and hydrophobic forces to form stable complexes. Circular dichroism showed that gallic acid and epigallocatechin gallate modified the secondary structure of lactoferrin by transiting from some α-helix to β-sheet. Based on molecular docking results, the binding energies of gallic acid-lactoferrin complex was -4.91 kcal/mol, and epigallocatechin gallate-lactoferrin complex was -4.72 kcal/mol. The minimum inhibitory concentration of gallic acid against E. coli was 4 mg/mL, epigallocatechin gallate against E. coli was 2 mg/mL, and LF against E. coli was 10 mg/mL. Fractional inhibitory concentration experiments indicated that the gallic acid-lactoferrin complex and epigallocatechin gallate-lactoferrin complex acted additively to inhibit E. coli. Scanning electron microscopy and crystal violet staining experiments showed that the complex inhibited the growth of E. coli by disrupting the cell membrane. Metabolomics analysis further indicated that these complexes disrupted the metabolism of E. coli and hindered protein synthesis. In conclusion, gallic acid and epigallocatechin gallate can complex with lactoferrin without compromising its antimicrobial activity and can be used in combination to inhibit the growth of pathogens such as E. coli.