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

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 hours 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 hours 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 A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Current Perspectives on Viable but Non-culturable State in Foodborne Pathogens

“…In general, VBNC cells are resistant to multiple antimicrobials, which may cause treatment failure at times (Hu and Coates, 2012). In fact, a recent study found that foods subjected to antimicrobial treatment harbored considerable numbers of VBNC cells (Anvarian et al, 2016). The presence of VBNC cells is considered a threat to human health and food safety due to the shortened shelf life that cause early spoilage of food products (Ayrapetyan and Oliver, 2016).…”

Use of flow cytometry and total viable count to determine the effects of orange juice composition on the physiology of Escherichia coli

“…This is indicative of other components of OJ having a protective effect on E. coli . Even when passed through a 1.2‐μm filter, OJ contains many compounds that might have a protective effect on E. coli (Anvarian et al., ). This phenomenon has been observed before in other studies comparing foodstuffs and model foodstuffs (Nualkaekul & Charalampopoulos, ; Uljas & Ingham, ).…”

“…E. coli K‐12 strain MG1655 was used due to its suitability as a surrogate strain for E. coli O157:H7 in food microbiological studies (Anvarian et al., ; Valdramidis, Geeraerd, & Van Impe, ). A single colony of E. coli was taken from a nutrient agar (Oxoid) plate inoculated into 20 ml of 2 × LB (Luria‐Bertani broth; 20 g/L tryptone, 10 g/L yeast extract, 10 g/L NaCl) in a 250‐mL conical flask and was grown for 18 hr at 37°C and 150 rpm shaking.…”

“…A BD Accuri C6 (BD, Oxford, UK) flow cytometer was used. Samples were diluted in PBS and stained with propidium iodide (PI; Sigma) and Bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol (DiBAC 4 (3) or BOX; Life Technologies) to determine viability and membrane potential, respectively (Anvarian et al., ). PI is a red nucleic acid dye that can only enter bacteria with a compromised inner membrane, signifying death.…”

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

“…The primary aim of this study was to use flow cytometry (FCM) to investigate the effects of washing E. coli K‐12 SCC1 with the sanitizers AC, H 2 O 2 , and organic acids on the viability, physiological state, and pH i of the cells as well as their culturability as measured using plate counts, before and after their inoculation in OJ. E. coli K‐12 has previously been used as a model for E. coli O157:H7 (Anvarian, Smith, & Overton, ; Valdramidis, Geeraerd, & Van Impe, ). We reveal the presence of VBNC bacteria in OJ following washing with both AC and H 2 O 2 , use real‐time FCM to determine the effects of acid and surfactant on available chlorine washing, and demonstrate the effects of organic acids on cells subsequently incubated in OJ.…”