A Fast Molecular Nondestructive Protocol for Evaluating Aerobic Bacterial Load on Fresh-Cut Lettuce

Abstract

Elaboration of minimally processed or fresh-cut vegetables requires a quick and reliable method for detection of bacterial contamination over the recommended limits. PCR-based methods fulfil these requirements, but amplification from DNA preparations of the food product is often hampered due to inhibiting substances. The purpose of this study was to develop a fast quantitative PCR (qPCR)-based method for aerobic bacterial enumeration in fresh-cut lettuce, using as reference the centrifugation water (CW) that comes up during processing instead of the food matrix itself. Comparisons between bacterial numbers on lettuce leaves before processing and bacterial numbers in the CW both for naturally occurring bacterial populations and for artificially inoculated lettuce were performed. On an average, 35% of the natural bacterial population and 64% of inoculated bacteria were recovered in the CW. Bacterial number in CW was proportional to initial lettuce contamination suggesting that measures on CW allow a narrow estimation of lettuce contamination. In qPCR, a 23S rDNA region was amplified from bacterial DNA present in the CW, followed by melting peak analyses and quantification. Enumeration of cell number by qPCR did not differ significantly from plate assay and might therefore replace it. The proposed protocol, which includes sample taking, DNA extraction and qPCR from the CW can be performed within less than 5 h. The resulting quantification might be used as a proxy of initial lettuce contamination, allowing direct intervention measures before fresh-cut commodity is shipped from the factory.

Keywords

minimally processed vegetables quantitative PCR microbial growth food safety

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