Purification, Characterization and Spectrofluorometric Study of Buffalo Lactoferrin Coupled to Amoxicillin

Antibiotic resistance raises concerns about their residues in food and the environment. To produce antibiotic-free dairy products, countries explore new strategies to reduce antibiotic doses in mastitis treatment. Lactoferrins (LF), iron-binding glycoproteins, exhibit species-specific glycosylation affecting their properties. While formulations of bovine lactoferrin have been tested with antibiotics showed synergistic properties increasing the antibacterial properties, other interactions of lactoferrin isoforms with antibiotics remain unclear. This study isolates, purifies, and characterizes buffalo lactoferrin (Bf-LF) and its interaction with amoxicillin. Bf-LF was purified using a Sephacryl S-100 column and monitored spectrofluorometrically. Spectroscopic analysis of Bf-LF interaction with amoxicillin reveals that increasing amoxicillin concentration reduces Bf-LF fluorescence, indicating conformational changes. The Stern-Volmer quenching constant (KSV) and binding constant (Ka) measured at 298 K shows values around (2.79 ± 0.29) × 104 L mol-1 and (0.300 ± 0.04) × 104 L mol−1, respectively. UV-Vis spectroscopy shows a red shift (258.08 to 271.83 nm) implying Bf-LF-amoxicillin binding. In silico studies suggest polar interactions between amoxicillin and specific Bf-LF amino acids.