Changes in spinach phylloepiphytic bacteria communities following minimal processing and refrigerated storage described using pyrosequencing of 16S rRNA amplicons.

AIMS: To investigate the changes in bacterial diversity on fresh spinach phyllosphere associated with storage at refrigeration temperatures. METHODS AND RESULTS: Community structure and population dynamics of spinach phylloepiphytic bacteria associated with packaging and refrigeration of ready-to-eat fresh produce were evaluated using pyrosequencing of 16S rRNA gene amplicons. A diverse community responsive to storage at refrigerated temperatures was detected belonging to over 1000 operational taxonomic units, including many diverse members not previously described on the phyllosphere. Of the approx. 8800 unique sequences examined from fresh spinach leaves, 75% were from previously undescribed taxa. The classified sequences from the fresh spinach phyllosphere were assigned to 11 different phyla with the largest number of reads belonging to Proteobacteria and Firmicutes. Packaging and storage of spinach under refrigerated conditions decreased the richness, diversity and evenness of the bacterial community. Refrigeration at 4 and 10°C and storage resulted in a decrease in number of taxa represented from 11 phyla in fresh spinach to only 5 phyla after 1 day of storage. Sequences belonging to γ-Proteobacteria, particularly Pseudomonas spp. and members of the Enterobacteriaceae, were the most numerous after 15 days of storage at both temperatures. Growth inhibition of the genera Escherichia was achieved at 4°C but not at 10°C storage, thus highlighting the importance of temperature in fresh packaged spinach. CONCLUSIONS: The application of pyrosequencing to describe composition and diversity of the phyllosphere on spinach leaves provided a broader outlook of the bacterial composition of this community complementing other phyllosphere studies that have used culture- and nonculture-dependent approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: Pyrosequencing allowed a broader description of the bacterial composition and diversity of the spinach leaf surface than previously obtained using culture-based detection and will be a powerful tool to help ensure the future safety and quality of packaged spinach.

Survival of Listeria monocytogenes, Listeria innocua, and lactic acid bacteria in chill brines.

Listeria monocytogenes is the pathogen of concern in ready-to-eat (RTE) meat products. Salt brines are used to chill processed meats. L. monocytogenes and lactic acid bacteria (LAB) can grow under saline conditions, and may compete with each other for nutrients. The objective of this study was to determine the effect of lactic acid bacteria (Enterococcus faecalis, Carnobacterium gallinarum, and Lactobacillus plantarum) on the survival of L. monocytogenes and Listeria innocua in brines stored under low temperatures for 10 d. Sterile tap water (STW) and 2 brine solutions (7.9% and 13.2% NaCl) were inoculated with 1 of 5 cocktails (L. monocytogenes, L. innocua, LAB, L. monocytogenes+ LAB, or L. innocua+ LAB) at initial concentrations of 7 log CFU/mL. Brines were stored for 10 d at 4 or 12 degrees C. Three replications of each brine concentration/cocktail/temperature combination were completed. No significant reductions of L. monocytogenes occurred in 7.9%[w/v] or 13.2%[w/v] brines when LAB were present; however, there were significant reductions after 10 d of L. monocytogenes in the STW solution when LAB were present (1.43 log CFU/mL at 4 degrees C and 3.02 log CFU/mL at 12 degrees C). L. innocua was significantly less resilient to environmental stresses of the brines than L. monocytogenes, both with and without LAB present (P< or = 0.05). These strains of lactic acid bacteria are not effective at reducing L. monocytogenes in brines at low temperatures. Furthermore, use of L. innocua as a model for L. monocytogenes is not appropriate under these environmental conditions.

Use of UV light for the inactivation of Listeria monocytogenes and lactic acid bacteria species in recirculated chill brines.

Ready-to-eat meat products have been implicated in several foodborne listeriosis outbreaks. Microbial contamination of these products can occur after thermal processing when products are chilled in salt brines. The objective of this study was to evaluate UV radiation on the inactivation of Listeria monocytogenes and lactic acid bacteria in a model brine chiller system. Two concentrations of brine (7.9% [wt/wt] or 13.2% [wt/wt]) were inoculated with a approximately 6.0 log CFU/ml cocktail of L. monocytogenes or lactic acid bacteria and passed through a UV treatment system for 60 min. Three replications of each bacteria-and-brine combination were performed and resulted in at least a 4.5-log reduction in microbial numbers in all treated brines after exposure to UV light. Bacterial populations were significantly reduced after 5 min of exposure to UV light in the model brine chiller compared with the control, which received no UV light exposure (P < 0.05). The maximum rate of inactivation for both microorganisms in treated brines occurred between minutes 1 and 15 of UV exposure. Results indicate that in-line treatment of chill brines with UV light reduces the number of L. monocytogenes and lactic acid bacteria.