Production of Recombinant Antimicrobial Polymeric Protein Beta Casein-E 50-52 and Its Antimicrobial Synergistic Effects Assessment with Thymol.

Shohreh Fahimirad,Seyed Razavi,Houshang Alizadeh,Hamid Abtahi,Mansour Ghorbanpour

doi:10.3390/molecules22060822

Abstract

Accelerating emergence of antimicrobial resistance among food pathogens and consumers’ increasing demands for preservative-free foods are two contemporary challenging aspects within the food industry. Antimicrobial packaging and the use of natural preservatives are promising solutions. In the present study, we used beta-casein—one of the primary self-assembly proteins in milk with a high polymeric film production capability—as a fusion partner for the recombinant expression of E 50-52 antimicrobial peptide in Escherichia coli. The pET21a-BCN-E 50-52 construct was transformed to E. coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg/mL fusion protein by ultrafiltration. Antimicrobial activities of recombinant BCN-E 50-52 performed against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus, and Candida albicans. Subsequently, the synergistic effects of BCN-E 50-52 and thymol were assayed. Results of checkerboard tests showed strong synergistic activity between two compounds. Time–kill and growth kinetic studies indicated a sharp reduction of cell viability during the first period of exposure, and SEM (scanning electron microscope) results validated the severe destructive effects of BCN E 50-52 and thymol in combination on bacterial cells.

Highlights

The adverse impacts of chemical antimicrobial compounds as food preservatives on human health, growing food pathogen resistance to commonly-used antimicrobial agents, and increasing interest in natural food preservatives are the primary issues persuading researchers to find novel natural antimicrobial compounds as food preservatives [1]
Bacteriocins are bacterial antimicrobial peptides which have been exploited as biopreservatives in the food industry for a hundred years [3]
BCN E 50-52 and thymol separately caused more than 50% reductions in the turbidity of both bacterial suspensions after 4 h and maintained this rate for 8 h (Figure 4)

Summary

Introduction

The adverse impacts of chemical antimicrobial compounds as food preservatives on human health, growing food pathogen resistance to commonly-used antimicrobial agents, and increasing interest in natural food preservatives are the primary issues persuading researchers to find novel natural antimicrobial compounds as food preservatives [1]. Antimicrobial packaging systems serve as a strong obstacle for microbial agents, and extend food shelf life while diminishing preservative compound utilization. Novel antimicrobial polymers with natural sources have received considerable attention [2]. Bacteriocins are bacterial antimicrobial peptides which have been exploited as biopreservatives in the food industry for a hundred years [3]. Bacteriocins have been divided into four classes: Class II consists of three subgroups on the basis of their primary structure.

Methods

Results

Conclusion