Susceptibility of the Different Oxygen-Sensing Probes to Interferences in Respirometric Bacterial Assays with Complex Media.

Respirometric microbial assays are gaining popularity, but their uptake is limited by the availability of optimal O2 sensing materials and the challenge of validating assays with complex real samples. We conducted a comparative evaluation of four different O2-sensing probes based on Pt-porphyrin phosphors in respirometric bacterial assays performed on standard time-resolved fluorescence reader. The macromolecular MitoXpress, nanoparticle NanO2 and small molecule PtGlc4 and PtPEG4 probes were assessed with E. coli cells in five growth media: nutrient broth (NB), McConkey (MC), Rapid Coliform ChromoSelect (RCC), M-Lauryl lauryl sulfate (MLS), and Minerals-Modified Glutamate (MMG) media. Respiration profiles of the cells were recorded and analyzed, along with densitometry profiles and quenching studies of individual media components. This revealed several limiting factors and interferences impacting assay performance, which include probe quenched lifetime, instrument temporal resolution, inner filter effects (mainly by indicator dyes), probe binding to lipophilic components, and dynamic and static quenching by media components. The study allowed for the ranking of the probes based on their ruggedness, resilience to interferences and overall performance in respirometric bacterial assays. The 'shielded' probe NanO2 outperformed the established MitoXpress probe and the small molecule probes PtGlc4 and PtPEG4.

Combining the oxygen sensor based respirometry and 16S rRNA amplicon sequencing for the analysis of microbiota in commercial mince products.

In this study, rapid respirometric microbial testing was combined with 16S rRNA amplicon sequencing, to assess the composition of microbiota in a total of 64 samples of commercial beef, turkey, lamb and pork mince. The O2 sensor-based respirometry system, while producing the anticipated total aerobic viable counts (TVC) data and patterns for most samples, also revealed unusual (linear) respiration profiles for some samples, mostly lamb and pork mince. The TVC values for beef mince, produced by respirometry and calculated using the available calibration equation, correlated well with the conventional plate counting method, ISO 4833-1:2013, 2013, while for the other species the correlation was less good. These effects, not observed in previous studies employing various food matrices, require further investigation. Using the same samples (crude homogenates) as in respirometry, the whole microbiome was also analysed by 16S rRNA amplicon sequencing for each mince-type. The sequencing showed an overall decrease in alpha diversity over shelf-life, with lamb and pork mince maintaining a proportion of rare taxa. Some taxa exhibited significant changes in abundance over shelf-life and after the respirometric analysis, with beef mince exhibiting a decrease in aerobic bacteria and an increase in facultative anaerobes. Beta diversity was also seen to depend on mince-type. Thus, the combined use of respirometry and sequencing techniques shows promise as a useful and unique analytical approach for food quality and safety evaluation, However, more data points and in-depth analysis are required to back up the findings of this initial study.

A sensor-based system for rapid on-site testing of microbial contamination in meat samples and carcasses.

AIMS: To develop an oxygen sensor-based method for testing total aerobic viable counts (TVC) in raw meat samples and cattle carcass swabs, which is rapid, simple, affordable, provides good sensitivity and analytical performance and allows on-site use. METHODS AND RESULTS: The test uses the same sample preparation procedure as the established plate counting TVC method for meat samples and carcasses, ISO4833-1:2013. After this liquid samples are transferred into standard 25-ml vials with built-in phosphorescent O2  sensors and incubated on a block heater with hourly readings of sensor signals with a handheld reader, to determine signal threshold time (TT, hours) for each sample. The method is demonstrated with the quantification of TVC in industrial cuts of raw beef meat (CFU per g) and carcass swabs (CFU per cm2 ). Calibration curves were generated, which give the following analytical equations for calculating the TVC load in unknown samples from measured TT values: TVC [Log(CFU per cm2 )] = 7.83-0.73*TT(h) and TVC [Log(CFU per g)] = 8.74-0.70*TT(h) for the carcass swabs and meat samples respectively. The new tests show good correlation with the ISO methods, with correlation coefficients 0.85 and 0.83 respectively. The testing requires no dilutions, covers the ranges 2-7 Log(CFU per g) for the meat samples and 1-7 Log(CFU per cm2 ) for carcass swabs, and has time to result 1-10 h with faster detection of more contaminated samples. CONCLUSIONS: The sensor-based testing demonstrates simplicity, high speed, sample throughput and automation. It can provide a straightforward replacement for the conventional TVC tests, which are time consuming, laborious and have time to result of 48-72 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The method(s) can be adopted by the meat industry and research labs, and used to improve microbial quality and safety of meat products and processes.

A Simple Sensor System for Onsite Monitoring of O2 in Vacuum-Packed Meats during the Shelf Life.

Vacuum packaging (VP) is used to reduce exposure of retail meat samples to ambient oxygen (O2) and preserve their quality. A simple sensor system produced from commercial components is described, which allows for non-destructive monitoring of the O2 concentration in VP raw meat samples. Disposable O2 sensor inserts were produced by spotting small aliquots of the cocktail of the Pt-benzoporphyrin dye and polystyrene in ethyl acetate onto pieces of a PVDF membrane and allowing them to air-dry. These sensor dots were placed on top of the beef cuts and vacuum-packed. A handheld reader, FirestinGO2, was used to read nondestructively the sensor phase shift signals (dphi°) and relate them to the O2 levels in packs (kPa or %). The system was validated under industrial settings at a meat processing plant to monitor O2 in VP meat over nine weeks of shelf life storage. The dphi° readings from individual batch-calibrated sensors were converted into the O2 concentration by applying the following calibration equation: O2 (%) = 0.034 * dphi°2 - 3.413 * dphi° + 85.02. In the VP meat samples, the O2 levels were seen to range between 0.12% and 0.27%, with the sensor dphi signals ranging from 44.03° to 56.02°. The DIY sensor system demonstrated ease of use on-site, fast measurement time, high sample throughput, low cost and flexibility.

Facile biosensor-based system for on-site quantification of total viable counts in food and environmental swabs.

We describe a new biosensor platform for rapid and simple quantification of total aerobic viable counts of bacteria (TVC) in food and environmental swabs by oxygen respirometry. The system uses disposable swab vials with phosphorescent oxygen sensors integrated in the bottom part, a small block heater/incubator and a handheld sensor reader. In the testing, groups of 1-20 swabs samples were prepared using the standard method (ISO, 18593:2018) in sensor vials, which were then incubated at 30 °C and measured hourly in a contactless, non-invasive manner. The measurements reveal time profiles of dissolved O2 in each sample vial, from which Threshold Time of sensor signal was determined and then TVC values (CFU/cm2) were calculated using the calibration equation. The method covers the range of 0.65-7.87 Log (CFU/cm2) and produces results in 1-8 hrs. The test was validated with swab samples from surfaces contaminated with E. coli, with whole meat microbiota, and with real environmental swabs. The results showed no statistically significant difference with the reference method which takes 48-72 h. The swab testing platform is fast and accurate, simple (sample-and-measure), portable, low cost (<$5k), requires no serial dilutions and is suitable for on-site deployment and use.