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

https://doi.org/10.1016/j.ijfoodmicro.2015.11.016Get rights and content

Highlights

  • The effect of clarification of orange juice on the culturability and physiology of Escherichia coli K-12 was tested.

  • Flow cytometry was used to rapidly detect changes in physiology and intracellular pH.

  • Clarification of orange juice alters both culturability and viability of E. coli.

Abstract

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 °C, a significantly lower number of healthy cells and a significantly higher number of injured cells were observed in 0.22 μm-filtered OJ at 24 h post-inoculation, compared to filtered OJ samples containing particles between 0.22 μm and 11 μm in size. Similarly, there was a significant difference between the number of healthy bacteria in the 0.7 μm-filtered OJ and both 0.22 μm-filtered and 1.2 μm-filtered OJ after 24 hour incubation at 22.5 °C. This indicated that OJ cloud between 0.7 μm and 0.22 μm 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 °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.

Introduction

When compared to conventionally-processed foods (such as pasteurised or UHT foods), many minimally-processed foods have more desirable organoleptic properties and greater nutritional value, and thus are of higher value for consumers (Pasha et al., 2014, Ragaert et al., 2004). However, elimination of harsh processing steps shortens shelf life by failing to eliminate spoilage microorganisms. As such, it is desirable to understand the effects of the composition of minimally processed foods on bacterial physiology, such that the food might be engineered to decrease growth of spoilage microorganisms and thus increase product shelf life.

Orange juice (OJ) is a foodstuff available in a range of forms, from highly processed to minimally-processed. One of the major production stages of OJ is clarification, intended to remove excess seeds, pieces of orange fruit and membrane as well as bitter essential oils such as limonene present in the freshly squeezed OJ (Rutledge, 1996). Depending on the OJ product, up to 12% (w/v) pulp is added back to the clarified juice before packaging (Berlinet et al., 2007). OJ can also be supplemented with homogenised pulp in order to meet the demand of the consumers for smoother OJ products (Sorenson and Bogue, 2003).

Flow cytometry (FCM) is a rapid technique that can be used to enumerate and determine optical and fluorescent properties of particles (Müller and Nebe-von-Caron, 2010). Although most commonly used for analysis of mammalian cells, it has many applications in microbiology and can be used to determine bacterial viability and physiology. An advantage of FCM over the total viable count (TVC) method is that it does not rely upon microbial growth for analysis, allowing detection of bacteria that are unable to grow on agar plates, the so-called VBNC (viable but non-culturable) phenotype (Oliver, 2005). Bacteria readily enter the VBNC state when exposed to many stresses. FCM has been used in some food processing applications (Comas-Riu and Rius, 2009), including analysis of apple juice (Yamaguchi et al., 2003), but it is often not well-suited to foods containing large numbers of particles of similar size to the bacteria under study. As such, pre-treatment is often required to remove particles of similar size to bacteria (such as colloidal fats and proteins in milk; Gunasekera et al., 2000) or additional techniques are required to differentiate bacteria from particles present in the food matrix, such as antibody staining (Clarke and Pinder, 1998) or the use of fluorescently-labelled bacteria. This also allows detection of lower numbers of bacteria using FCM methods.

OJ contains many compounds with antimicrobial activities such as essential oils, primarily limonene (Fernández-Vázquez et al., 2013) and flavanones, primarily hesperidin (Bisignano and Saija, 2002, Di Pasqua et al., 2007, Garg et al., 2001). It has also been shown that the majority of these compounds, especially the limonene and hesperidin, are mainly present in the pulp (defined as particulate matter > 2 μm in size; Brat et al., 2003) and cloud (particulates < 2 μm in size) of OJ (Ben-Shalom and Pinto, 1999, Brat et al., 2003). As a result, it was hypothesised that OJ clarification and the resultant removal of these compounds could lead to improved survival of microbes in OJ and thereby an alteration of the microbiological risk posed by OJ products of different formulations (Sampedro et al., 2011). The main aims of the current study were to test this hypothesis, and to investigate the utility of FCM as a technique of monitoring physiology of bacteria in orange juice. The effects of OJ filtration on the physiology of E. coli K-12 MG1655 (an enteric marker strain; Valdramidis et al., 2007) were determined using TVC, indicating the viability of bacteria as determined by their ability to grow on agar plates, and FCM using the dye propidium iodide (PI) which stains dead E. coli (Shi et al., 2007) indicating viability in a non-growth dependent manner. Although having different acid tolerance characteristics to pathogenic E. coli strains, E. coli K-12 is very well-characterised in terms of stress responses and physiology. FCM was also used to determine membrane potential and intracellular pH (pHi) of bacteria in OJ. As far as the authors are aware, this is the first study of the effects of OJ clarification on bacterial viability or physiology.

Section snippets

OJ clarification and filtration

Freshly-squeezed OJ was obtained from a local retailer and centrifuged at 17,696 g for 40 min to remove pulp. The supernatant (pulp-free OJ) was then filtered through sterile filter papers with pore sizes of 11 μm, 8 μm, 1.6 μm or 1.2 μm. The 1.2 μm-filtered OJ was also then filtered through sterile 0.7 μm filter paper or 0.22 μm syringe filters.

Particle size distribution

The size distribution of cloud particles in OJ was measured by laser diffraction using a Malvern Mastersizer 2000 equipped with a Malvern Hydro 2000SM particle

Characterisation of OJ cloud particles

In order to clarify OJ using filtration, the pulp was first removed by centrifugation. Pulp-free OJ was then filtered using filters with different pore sizes, and the particle size distribution of the resultant filtered OJ samples analysed by Mastersizer (Supplementary Fig. 1a). Following filtration, the filter papers were also visually examined (Supplementary Fig 1b). Depending on the filtration regime, three cloud populations of descending size could be observed. First, large particles of

Conclusion

FCM has been demonstrated to be a technique amenable to investigation of the physiology of E. coli in OJ. Although not the most common infectious agent present in OJ, E. coli has previously been reported as causing outbreaks of OJ-related foodborne disease (Singh et al., 1995) and the interaction of E. coli and orange fruit has also been investigated (Eblen et al., 2004).

Clarification of OJ by centrifugation and filtration was shown to have significant effects on the viability and physiology of

Acknowledgements

This work was supported by a UK Biotechnology and Biological Sciences Research Council PhD studentship to AA. The BD Accuri C6 flow cytometer was awarded to TWO by the BD Accuri Creativity Award. TWO and AA were paid speaker expenses by BD for speaking at BD Accuri users' events.

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