Flow cytometry and growth-based analysis of the effects of fruit sanitation on the physiology of Escherichia coli in orange juice

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Colleges, School and Institutes


Chlorine-based solutions are commonly used to sanitize orange fruits prior to juice extraction. We used flow cytometry (FCM) to investigate the physiology of Escherichia coli following its subjection to chlorine-based solutions and alternative sanitizing agents (H2O2 and organic acids). Green fluorescent protein (GFP)-generating E. coli K-12 were washed with 50-200 ppm available chlorine (AC), 1-5% H2O2, 2-4% citric acid, 4% acetic acid or 4% lactic acid, after which they were added to 1.2 μm-filtered orange juice (OJ). Cell physiology was investigated with FCM during storage at 4 ºC, and culturability was determined using plate counting. Analysis of GFP fluorescence allowed estimation of intracellular pH (pHi). FCM results demonstrated an inverse relationship between the concentration of AC or H2O2 and cellular health in OJ. Higher concentrations of sanitizer also resulted in a significantly greater number of viable but non-culturable (VBNC) cells. Real-time FCM showed that supplementation of AC with 2% citric acid, but not with 100 ppm of Tween-80, led to a significant reduction in pHi of the cells incubated in OJ, and that the majority of the reduction in pHi occurred during the first 2 minutes of incubation in OJ. Organic acids were found to be more effective than both AC and H2O2 in reducing the pHi, viability and culturability of the cells in OJ. The results confirmed the hypothesis that consecutive subjection of E. coli to maximum legally permitted concentrations of sanitizers and OJ induces the VBNC state. Furthermore, we demonstrate successful application of FCM for monitoring the efficacy of washing procedures.


Original languageEnglish
Pages (from-to)1072-1083
Number of pages12
JournalFood Science and Nutrition
Issue number3
Publication statusPublished - 7 Feb 2019


  • Escherichia coli, VBNC cells, Flow cytometry, Fruit sanitation, Orange juice

ASJC Scopus subject areas