The effects of orange juice clarification on the physiology of Escherichia coli; growth-based and flow cytometric analysis

摘要

Orange juice (OJ) is a food product available in various forms which can be processed to a greater or lesser extent. Minimally-processed OJ has a high consumer perception but presents a potential microbiological risk due to acid tolerant bacteria. Clarification of OJ (such as removal of cloud) is a common processing step in many OJ products. However, many of the antimicrobial components of OJ such as essential oils are present in the cloud fraction. Here, the effect of clarification by filtration on the viability and physiology of Escherichia coli K-12 was tested using total viable count (TVC) and flow cytometric (FCM) analysis. The latter technique was also used to monitor intracellular pH during incubation in OJ. Removal of the OJ cloud fraction was shown to have dramatic effects on bacterial viability and physiology during storage at a range of incubation temperatures. For instance, at 4 degrees C, a significantly lower number of healthy cells and a significantly higher number of injured cells were observed in 0.22 pm-filtered OJ at 24 h post-inoculation, compared to filtered OJ samples containing particles between 0.22 pm and 11 pm in size. Similarly, there was a significant difference between the number of healthy bacteria in the 0.7 pm-filtered OJ and both 0.22 gm-filtered and 12 pm-filtered OJ after 24 hour incubation at 22.5 degrees C. This indicated that OJ cloud between 0.7 pm and 022 pm in size might have an adverse effect on the viability of E.coli K-12. Furthermore, FCM allowed the rapid analysis of bacterial physiology without the requirement for growth on agar plates, and revealed the extent of the viable but non-culturable (VBNC) population. For example, at 4 degrees C, while the FCM viable count did not substantially decrease until 48 h, decreases in TVC were observed between 0 and 48 hour incubation, due to a subset of injured bacteria entering the VBNC state, hence being unable to grow on agar plates. This study highlights the application of FCM in monitoring bacterial physiology in foods, and potential effects of OJ clarification on bacterial physiology. (C) 2015 Elsevier B.V. All rights reserved.