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Published on: October 1, 2015
Human lactoferrin from breast milk: characterization by HPLC and its in vitro antibiofilm performance
Karina Jeanette Parra-Saavedra1, Adriana Macaria Macias-Lamas1, Jorge Manuel Silva-Jara1
1Departamento de Farmacobiología, Centro Universitario de Ciencias Exactas e Ingenierías, Universidad de Guadalajara., Blvd. Marcelino García Barragán 1421, Olímpica, C.P. 44430 Guadalajara, Jalisco Mexico.
Insights
Human lactoferrin (hLf) purified from breast milk shows potential in preventing infections in preterm infants. This study details a method for hLf purification and demonstrates its significant antibiofilm activity against Pseudomonas aeruginosa.
Area of Science:
- Biochemistry
- Neonatal Medicine
- Microbiology
Background:
- Preterm infants face high infection risks from bacteria like Pseudomonas aeruginosa in NICUs.
- Human lactoferrin (hLf), abundant in breast milk, offers protection against neonatal infections.
Purpose of the Study:
- To develop a strategy for obtaining purified, lyophilized hLf from breast milk.
- To establish a reliable HPLC method for quantifying hLf.
- To evaluate the antibiofilm efficacy of purified hLf against P. aeruginosa.
Main Methods:
- hLf purification using heparin affinity chromatography and ultrafiltration (30 kDa membrane).
- Lyophilization of purified hLf.
- HPLC for hLf quantification (0.040-0.140 mg/mL range).
- FTIR spectroscopy and SDS-PAGE for molecular characterization.
- Biofilm inhibition assays against P. aeruginosa.
Main Results:
- A method yielding lyophilized hLf with approximately 70% recovery was established.
- The HPLC method demonstrated selectivity, accuracy, and repeatability.
- Purified hLf inhibited P. aeruginosa biofilm formation by 75% at a concentration of 6 mg/mL.
Conclusions:
- Lyophilized human lactoferrin can be effectively purified from breast milk.
- Purified hLf exhibits significant antibiofilm activity against Pseudomonas aeruginosa, suggesting its therapeutic potential for neonatal infections.
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.
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