Investigating the effect of lactoferrin loaded on chitosan against Pseudomonasaeruginosa

Background and Objective: The occurrence of increased resistance in bacterial strains to antimicrobialcompounds is considered one of the most important therapeutic problems worldwide. Pseudomonasaeruginosa is one of the most well-known opportunistic bacteria resistant to antibiotics, which plays animportant role in the occurrence of severe infections in the skin (especially in burns), and the respiratorysystem and blood circulation (۷% bacteremia). Several studies show that chitosan is a naturalmucopolysaccharide compound with antimicrobial properties. This compound is actually a non-toxic,biocompatible, and active cationic polymer that is derived from glucan with repeating chitin units andprevents the growth of bacteria. Also, lactoferrin is an iron-containing protein in milk, which, in additionto its anti-cancer, anti-inflammatory, and antioxidant properties, also has very good antimicrobial andantiviral activities. The antimicrobial mechanism of lactoferrin against gram-negative bacteria can be dueto their direct interaction with the lipid A part of lipopolysaccharide. As a result, by damaging the outermembrane and changing its permeability, lipopolysaccharide is released and the bacteria die. Therefore,due to the importance of the antimicrobial properties of the two mentioned compounds, the aim of thisresearch is to investigate the antimicrobial effect of camel milk lactoferrin twins loaded on chitosanagainst Pseudomonas aeruginosa (ATCC۲۷۸۵۳).Methods: In this research, after removing casein by ion exchange chromatography, CM Sephadex C-۵۰was used to purify lactoferrin from camel milk. In order to confirm the purification step, SDS-PAGEmethods and the absence of color in the presence of tetramethylbenzidine were used. Then, by dialysismethod in the presence of ۱۰ mM ammonium phosphate, the obtained product was concentrated and thedesired concentrations were prepared. In the next step, ۱% chitosan with medium molecular weight wasprepared with sterile distilled water and used after pH adjustment. Finally, the loading step of purifiedlactoferrin on chitosan was performed using TPP (thiamine pyrophosphate). To investigate theantimicrobial effect of lactoferrin (two concentrations of ۳۰۰ and ۳۵۰ μg/ml) loaded on ۱% chitosan, themicroassay method was used along with positive and negative controls.Findings: The results show that in the presence of a concentration of ۳۰۰ μg/ml of lactoferrin loaded onchitosan, a significant decrease (P-value < ۰.۰۱) was observed in the growth of Pseudomonas aeruginosacompared to the control group. ۳۵۰ μg/ml loaded lactoferrin also reported a significant decrease (P-value< ۰.۰۰۱).Conclusion: According to the obtained findings, it can be concluded that by loading lactoferrin onchitosan, their antimicrobial effect increases. Therefore, by using appropriate concentrations of lactoferrin(such as ۳۵۰ μg/ml) loaded on ۱% chitosan, a further reduction in the growth of Pseudomonas aeruginosawill be observed.

Keywords:

Antimicrobial properties , Chitosan , Lactoferrin , Loading , Pseudomonas aeruginosa