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
The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility.
The aim of this study was to compare approaches commonly recommended in the literature for the improvement of the survival of probiotics in the human digestive tract. The survival of two probiotics, Lactobacillus casei and Bifi dobacterium lactis, in the presence or absence of prebiotics, maize starch, fermented milk and upon encapsulation in calcium alginate-chitosan was evaluated. While B. lactis was resistant to stomach juice, but sensitive to duodenal juice, L. casei showed an exactly opposite behaviour. In contrast to other published studies, here the overall digestive survivability of probiotics was not improved signifi cantly by prebiotics, maize starch or encapsulation. However, a signifi cant improvement of the overall survivability of B. lactis (but not L. casei) during in vitro digestion was noted in milk and fermented milk, possibly due to reduction of the activity of bile against this probiotic. Summarising, no one method could be universally recommended for the improvement of probiotic survivability. Nevertheless, this research indicated that certain probiotic characteristics, such as susceptibility to bile or acid or ability to utilise matrix components as an energy source, could be used in further research to select the most effective approaches to deliver viable cells into lower parts of the digestive tract.
Orange juice (OJ) contains numerous compounds some of which are known to play key roles in growth and survival of bacteria. This study aimed to investigate the effects of natural or processing‐induced variations in OJ composition on the physiology of Escherichia coli. OJ and model OJ (MOJ) samples containing various sugars, organic acids, amino acids, or ascorbic acid were inoculated with E. coli K‐12 MG1655 in different growth phases. The culturability, viability, and physiology of the cells were investigated during storage using plate counting and flow cytometry. Generally, stationary‐phase cells displayed the greatest survival in both MOJ and OJ. Increase in incubation temperature from 4 to 22.5ºC caused a significant decrease in both healthy and culturable cell populations. Supplementation of MOJ with ascorbic acid and amino acids increased both the viability and culturability of the cells. Similar trends were observed in amino acid‐supplemented OJ, albeit at a slower rate. In contrast, variations in sugar or organic acid composition had negligible effects on the physiological status of the cells. In summary, natural variation in ascorbic acid or amino acid concentrations could potentially have an adverse effect on the microbiological safety of orange juice.
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 (H 2 O 2 and organic acids). Green fluorescent protein ( GFP )‐generating E. coli K‐12 were washed with 50–200 ppm available chlorine ( AC ), 1%–5% H 2 O 2 , 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°C, and culturability was determined using plate counting. Analysis of GFP fluorescence allowed estimation of intracellular pH ( pH i ). FCM results demonstrated an inverse relationship between the concentration of AC or H 2 O 2 and cellular health in OJ . Higher concentrations of sanitizer also resulted in a significantly greater number of viable but nonculturable ( 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 pH i of the cells incubated in OJ , and that the majority of the reduction in pH i occurred during the first 2 min of incubation in OJ . Organic acids were found to be more effective than both AC and H 2 O 2 in reducing the pH i , 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.
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