Quantification of Carnobacterium maltaromaticum Bacteriocin Gene Expression on Refrigerated Vacuum-packaged Ham

Introduction: Carnobacterium maltaromaticum is a bacteriocin-producing protective culture approved for use in food. Bacteriocins produced by C. maltaromaticum ATCC^®PTA-5313 inhibit the growth of Listeria monocytogenes; when used in combination with other antimicrobials, L. monocytogenes on ready-to-eat meats is killed to levels below the detection limit. C. maltaromaticum produces three different bacteriocins, carnocyclin, piscicolin 126 and carnobacteriocin BM1; however, it remains unclear which of the three compounds is responsible for the preservative effect on meat. 

Purpose: The aim of this study was to quantify the gene expression of the three bacteriocins produced by C. maltaromaticum ATCC^®PTA-5313 on vacuum packaged ham stored at 4°C. 

Methods: Ham was inoculated with 10³ CFU/cm² C. maltaromaticum ATCC^® PTA-5313, vacuum packaged and stored at 4°C for up to 28 days. Samples were taken throughout storage for quantification of cell counts, and for RNA isolation. RNA was extracted using the Qiagen RNeasy kit and followed by reverse transcription. Quantitative Real Time Polymerase Chain Reaction was used to determine expression of the structural genes for carnocyclin A (cclA), piscicolin 126 (pisA), carnobacteriocin BM1 (cbnBM1) and housekeeping gene 16SrRNA.  Data from three replicates was analyzed using the comparative C_T method.  

Results: The expression of cclA, pisA, and cbnBM1 was quantified from refrigerated ham. Cell counts indicated that C. maltaromaticum ATCC^® PTA-5313 was present at levels between 6-8 log CFU/cm² when RNA was extracted. C_T values lower than 35 were considered positive for expression of the bacteriocins. C_Tvalues on day 21 and 28 for carnocyclin A were between 21.24 to 24.44, piscicolin 126 ranged from 22.8 to 31.6 and carnobacteriocin BM1 ranged 23.4 to 27.1. 

Significance: This method could be used to confirm the production of bacteriocins by C. maltaromaticum ATCC^® PTA-5313 on ready-to-eat meats to control the growth of L. monocytogenes.