Human lactoferrin from breast milk: characterization by HPLC and its in vitro antibiofilm performance

Abstract

Preterm infants are at high risk of infection due to opportunistic bacteria as Pseudomonas aeruginosa, causing infections among infants in neonatal intensive care units. Human lactoferrin (hLf) is a multifunctional protein and one of the most abundant in breast milk, and plays an important role in prevention of different infections in neonates. This work offers a strategy to obtain a lyophilisate of purified lactoferrin from breast milk. In addition, a reliable HPLC method for quantification of lactoferrin with a linear quantification range of 0.040-0.140 mg/mL with selectivity, accuracy and repeatability, is described. Lyophilized hLf was obtained by purification through a heparin affinity column followed by ultrafiltration with a 30 kDa membrane. The final solution was lyophilized and the product was analyzed using HPLC method, recovering about 70% of initial lactoferrin in the sample. This molecule was elucidated through FTIR spectroscopy and SDS-PAGE electrophoresis. In addition, the capacity against biofilm formation of P. aeruginosa was demonstrated with 75% of inhibition at 6 mg/mL. These results suggest that lyophilized hLf can be obtained by purification of breast milk and that it can provide antibiofilm activity against P. aeruginosa.

Keywords: Antibiofilm activity; HPLC; Heparin column; Human lactoferrin; Lyophilized lactoferrin.

Fig. 1

Chromatograms of. A Ultrapasteurized milk used as sample blank, B hLf STD, and C Breast milk sample. There is no interference present at the same retention time as the hLf

Fig. 2

FT-IR spectra of hLf STD and lyophilized lactoferrin. The spectrum of the sample has the same pattern of signals as for the standard, confirming the identity of the protein in the lyophilisate. The dotted lines show the most representative signals for the identification of lactoferrin

Fig. 3

SDS-PAGE gel of hLf standard and lyophilized lactoferrin. The band of greatest intensity around 80 kDa corresponds to the molecular weight of lactoferrin. This band is present in the standard and the sample

Fig. 4

Antibiofilm activity of lyophilized lactoferrin at various concentrations against Pseudomonas aeruginosa. Biofilm formation was measured after 24 h of incubation of bacteria with different concentrations of lyophilized lactoferrin. Bars represent the percentage of inhibition of biofilm, while the optical density of biofilm with the blue line is represented. The maximum antibiofilm activity was obtained at 6 mg/mL with 75% inhibition of biofilm formation. In addition, an inverse relationship between the percentage of inhibition and the optical density obtained for each concentration of the experiment is observed. Different letters show statistical significant differences (p < 0.05) by Tukey’s test