Abbas Tanhaeian1,2, Mehdi Mirzaii2, Zana Pirkhezranian3, Mohammad Hadi Sekhavati4. 1. Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, P.O. Box 91775-1163, Mashhad, Iran. 2. School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran. 3. Department of Animal Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad, P.O. Box 91775-1163, Mashhad, Iran. 4. Department of Animal Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad, P.O. Box 91775-1163, Mashhad, Iran. Sekhavati@um.ac.ir.
Abstract
BACKGROUND: Foodborne pathogens and their biofilms are considered as one of the most serious problems in human health and food industry. Moreover, safety of foods is a main global concern because of the increasing use of chemical food additives. Ensuring food safety enhances interest in discovery of new alternative compounds such as antimicrobial peptides (AMPs), which can be used as bio-preservatives in the food industry. In this study, the most important antimicrobial peptides of camel milk lactoferrin (lactoferrampin and lactoferricin) were recombinantly expressed in the form of chimeric peptide (cLFchimera) in a food-grade L. lactis strain. P170 expression system was used to express secreted cLFchimera using pAMJ1653 expression vector which harbors a safe (non-antibiotic) selectable marker. RESULTS: Peptide purification was carried out using Ni-NTA agarose column from culture medium with concentration of 0.13 mg/mL. The results of disk diffusion test revealed that cLFchimera had considerable antimicrobial activity against a number of major foodborne bacteria. Furthermore, this chimeric peptide showed strong and weak inhibitory effect on biofilm formation against P. aeruginosa, S. aureus E. faecalis, and E. coli, respectively. Antioxidant activity and thermal stability of the chimeric peptide was determined. The results showed that cLFchimera had antioxidant activity (IC50: 310 μ/mL) and its activity was not affected after 40 min of boiling. Finally, we evaluated the interaction of the peptide with LPS and DNA in bacteria using molecular dynamic simulation as two main intra and extra cellular targets for AMPs, respectively. Our in silico analysis showed that cLFchimera had strong affinity to both of these targets by positive charged residues after 50 ns molecular dynamic simulation. CONCLUSIONS: Overall, the engineered food-grade L. lactis generated in the present study successfully expressed a secreted chimeric peptide with antimicrobial properties and could be considered as a promising bio-preservative in the food industry.
BACKGROUND: Foodborne pathogens and their biofilms are considered as one of the most serious problems in human health and food industry. Moreover, safety of foods is a main global concern because of the increasing use of chemical food additives. Ensuring food safety enhances interest in discovery of new alternative compounds such as antimicrobial peptides (AMPs), which can be used as bio-preservatives in the food industry. In this study, the most important antimicrobial peptides of camel milk lactoferrin (lactoferrampin and lactoferricin) were recombinantly expressed in the form of chimeric peptide (cLFchimera) in a food-grade L. lactis strain. P170 expression system was used to express secreted cLFchimera using pAMJ1653 expression vector which harbors a safe (non-antibiotic) selectable marker. RESULTS: Peptide purification was carried out using Ni-NTAagarose column from culture medium with concentration of 0.13 mg/mL. The results of disk diffusion test revealed that cLFchimera had considerable antimicrobial activity against a number of major foodborne bacteria. Furthermore, this chimeric peptide showed strong and weak inhibitory effect on biofilm formation against P. aeruginosa, S. aureusE. faecalis, and E. coli, respectively. Antioxidant activity and thermal stability of the chimeric peptide was determined. The results showed that cLFchimera had antioxidant activity (IC50: 310 μ/mL) and its activity was not affected after 40 min of boiling. Finally, we evaluated the interaction of the peptide with LPS and DNA in bacteria using molecular dynamic simulation as two main intra and extra cellular targets for AMPs, respectively. Our in silico analysis showed that cLFchimera had strong affinity to both of these targets by positive charged residues after 50 ns molecular dynamic simulation. CONCLUSIONS: Overall, the engineered food-grade L. lactis generated in the present study successfully expressed a secreted chimeric peptide with antimicrobial properties and could be considered as a promising bio-preservative in the food industry.
Authors: Ali Daneshmand; Hassan Kermanshahi; Mohammad Hadi Sekhavati; Ali Javadmanesh; Monireh Ahmadian; Marzieh Alizadeh; Ahmed Aldawoodi Journal: Sci Rep Date: 2020-10-19 Impact factor: 4.379