Biochemical and Molecular Analysis of the Antilisterial Peptides Produced by Enterococcus hirae Strains Isolated from Raw Ewe Milk

Abstract

Aims: This study was conducted to identify and characterize the anti-listerial bacteriocins produced by Enterococcus hirae strains isolated from ewe milk. Study Design: Bacteriocins produced by E. hirae strains were identified and characterized by physio-chemical methods. Bacteriocin structural genes were evaluated by molecular methods. Place and Duration of Study: Biotechnology Department, Razi vaccine and Serum Research institute, and National institute of Animal Science, Iran, between January 2013 and March 2015. Methodology: Two E. hirae were isolated from raw ewe milk samples collected from Yengi Esperan (Sfeedan) village located in East Azerbaijan Province, Iran. The isolates demonstrating antilisterial activity were identified by 16S rRNA genes sequencing. The bacteriocinogenic potential of the isolates was evaluated using biochemical tests. The proteinaceous compounds were purified using Ammonium sulphate precipitation (40%), cation-exchange chromatography followed by SDS-PAGE analysis. Occurrence of enterocin structural genes was evaluated using a set of primers in a PCR reaction. Results: The antilisterial compounds produced by the two E. hirae strains were sensitive to the proteolytic enzymes, while catalase and lipase had no effect on the activity. In contrast to the bacteriocin Eh512, enterocin Eh514 showed partial sensitivity to the enzyme lysozyme. The proteinaceous agent from the two producer isolates; Eh512 and Eh514 were single peptides of approximately 6.5 and 5.8 KDa, respectively. The enterocins in study appeared heat stable and resistant to acidic pH values. Analysis of the enterocin structural genes, showed the presence of entA, and entB genes in both the isolates whereas, E. hirae Eh512 additionally harbored entP and entQ genes. Sequence analysis of entA genes in both isolates indicated 95% homology with other entA genes in NCBI library. Conclusion: The studied enterocins might be suitable replacement for chemical additives used in food preservations. However, further studies are required to validate these findings.