Development and assessment of new sensor systems in food packaging applications

The use of optical sensor technology for non-invasive determination of key quality pack parameters improved package/product quality. This technology can be used for optimization of packaging processes, improvement of product shelf-life and maintenance of quality. In recent years, there has been a major focus on O2 and CO2 sensor development as these are key gases used in modified atmosphere packaging (MAP) of food. The first and second experimental chapters (chapter 2 and 3) describe the development of O2, pH and CO2 solid state sensors and its (potential) use for food packaging applications. A dual-analyte sensor for dissolved O2 and pH with one bi-functional reporter dye (meso-substituted Pd- or Ptporphyrin) embedded in plasticized PVC membrane was developed in chapter 2. The developed CO2 sensor in chapter 3 was comprised of a phosphorescent reporter dye Pt(II)- tetrakis(pentafluorophenyl) porphyrin (PtTFPP) and a colourimetric pH indicator α-naphtholphthalein (NP) incorporated in a plastic matrix together with a phase transfer agent tetraoctyl- or cetyltrimethylammonium hydroxide (TOA-OH or CTA-OH). The third experimental chapter, chapter 4, described the development of liquid O2 sensors for rapid microbiological determination which are important for improvement and assurance of food safety systems. This automated screening assay produced characteristic profiles with a sharp increase in fluorescence above the baseline level at a certain threshold time (TT) which can be correlated with their initial microbial load and was applied to various raw fish and horticultural samples. Chapter 5, the fourth experimental chapter, reported upon the successful application of developed O2 and CO2 sensors for quality assessment of MAP mushrooms during storage for 7 days at 4°C.

Use of oxygen sensors for the non destructive measurement of oxygen in packaged food and beverage products and its impact on product quality and shelf life

The principal objective of this thesis was to investigate the ability of reversible optical O2 sensors to be incorporated into food/beverage packaging systems to continuously monitor O2 levels in a non-destructive manner immediately postpackaging and over time. Residual levels of O2 present in packs can negatively affect product quality and subsequently, product shelf-life, especially for O2-sensitive foods/beverages. Therefore, the ability of O2 sensors to continuously monitor O2 levels present within food/beverage packages was considered commercially relevant in terms of identifying the consequences of residual O2 on product safety and quality over time. Research commenced with the development of a novel range of O2 sensors based on phosphorescent platinum and palladium octaethylporphyrin-ketones (OEPk) in nano-porous high density polyethylene (HDPE), polypropylene (PP) polytetrafluoroethylene (PTFE) polymer supports. Sensors were calibrated over a temperature range of -10°C to +40°C and deemed suitable for food and beverage packaging applications. This sensor technology was used and demonstrated itself effective in determining failures in packaging containment. This was clearly demonstrated in the packaging of cheese string products. The sensor technology was also assessed across a wide range of packaged products; beer, ready-to-eat salad products, bread and convenience-style, muscle-based processed food products. The O2 sensor technology performed extremely well within all packaging systems. The sensor technology adequately detected O2 levels in; beer bottles prior to and following pasteurisation, modified atmosphere (MA) packs of ready-to-eat salad packs as respiration progressed during product storage and MA packs of bread and convenience-style muscle-based products as mycological growth occurred in food packs over time in the presence and absence of ethanol emitters. The use of the technology, in conjunction with standard food quality assessment techniques, showed remarkable usefulness in determining the impact of actual levels of O2 on specific quality attributes. The O2 sensing probe was modified, miniaturised and automated to screen for the determination of total aerobic viable counts (TVC) in several fish species samples. The test showed good correlation with conventional TVC test (ISO:4833:2003), analytical performance and ruggedness with respect to variation of key assay parameters (probe concentration and pipetting volume). Overall, the respirometric fish TVC test was simple to use, possessed a dynamic microbial range (104-107 cfu/g sample), had an accuracy of +/- one log(cfu/g sample) and was rapid. Its ability to assess highly perishable products such as fish for total microbial growth in <12 hr demonstrates commercial potential.

Multi-parametric metabolic assessment of cells under stress conditions

Glycolysis, glutaminolysis, the Krebs cycle and oxidative phosphorylation are the main metabolic pathways. Exposing cells to key metabolic substrates (glucose, glutamine and pyruvate); investigation of the contribution of substrates in stress conditions such as uncoupling and hypoxia was conducted. Glycolysis, O2 consumption, O2 and ATP levels and hypoxia inducible factor (HIF) signalling in PC12 cells were investigated. Upon uncoupling with FCCP mitochondria were depolarised similarly in all cases, but a strong increase in respiration was only seen in the cells fed on glutamine with either glucose or pyruvate. Inhibition of glutaminolysis reversed the glutamine dependant effect. Differential regulation of the respiratory response to FCCP by metabolic environment suggests mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function. At reduced O2 availability (4 % and 0 % O2), cell bioenergetics and local oxygenation varied depending on the substrate composition. Results indicate that both supply and utilisation of key metabolic substrates can affect the pattern of HIF-1/2α accumulation by differentially regulating iO2¬, ATP levels and Akt/Erk/AMPK pathways. Inhibition of key metabolic pathways can modulate HIF regulatory pathways, metabolic responses and survival of cancer cells in hypoxia. Hypoxia leads to transcriptional activation, by HIF, of pyruvate dehydrogenase (PDH) kinase which phosphorylates and inhibits PDH, a mitochondrial enzyme that converts pyruvate into acetyl-CoA. The levels of PDH (total and phosphorylated), PDH kinase and HIF-1α were analysed in HCT116 and HCT116 SCO2-/- (deficient in complex IV of the respiratory chain) grown under 20.9 % and 3 % O2. Data indicate that regulation of PDH can occur in a manner independent of the HIF-1/PDH kinase 1 axis, mitochondrial respiration and the demand for acetyl-CoA. Collectively these results can be applied to many diseases; reduced nutrient supply and O2 during ischemia/stroke, hypoglycaemia in diabetes mellitus and cancer associated changes in uncoupling protein expression levels.

New phosphorescence based probes and techniques for the analysis of cellular oxygen and respiration

Real time monitoring of oxygenation and respiration is on the cutting edge of bioanalysis, including studies of cell metabolism, bioenergetics, mitochondrial function and drug toxicity. This thesis presents the development and evaluation of new luminescent probes and techniques for intracellular O2 sensing and imaging. A new oxygen consumption rate (OCR) platform based on the commercial microfluidic perfusion channel μ-slides compatible with extra- and intracellular O2 sensitive probes, different cell lines and measurement conditions was developed. The design of semi-closed channels allowed cell treatments, multiplexing with other assays and two-fold higher sensitivity to compare with microtiter plate. We compared three common OCR platforms: hermetically sealed quartz cuvettes for absolute OCRs, partially sealed with mineral oil 96-WPs for relative OCRs, and open 96-WPs for local cell oxygenation. Both 96-WP platforms were calibrated against absolute OCR platform with MEF cell line, phosphorescent O2 probe MitoXpress-Intra and time-resolved fluorescence reader. Found correlations allow tracing of cell respiration over time in a high throughput format with the possibility of cell stimulation and of changing measurement conditions. A new multimodal intracellular O2 probe, based on the phosphorescent reporter dye PtTFPP, fluorescent FRET donor and two-photon antennae PFO and cationic nanoparticles RL-100 was described. This probe, called MM2, possesses high brightness, photo- and chemical stability, low toxicity, efficient cell staining and high-resolution intracellular O2 imaging with 2D and 3D cell cultures in intensity, ratiometric and lifetime-based modalities with luminescence readers and FLIM microscopes. Extended range of O2 sensitive probes was designed and studied in order to optimize their spectral characteristics and intracellular targeting, using different NPs materials, delivery vectors, ratiometric pairs and IR dyes. The presented improvements provide useful tool for high sensitive monitoring and imaging of intracellular O2 in different measurement formats with wide range of physiological applications.

An assessment of the influence of the industry distribution chain on the oxygen levels in commercial modified atmosphere packaged cheddar cheese using non-destructive oxygen sensor technology

The establishment and control of oxygen levels in packs of oxygen-sensitive food products such as cheese is imperative in order to maintain product quality over a determined shelf life. Oxygen sensors quantify oxygen concentrations within packaging using a reversible optical measurement process, and this non-destructive nature ensures the entire supply chain can be monitored and can assist in pinpointing negative issues pertaining to product packaging. This study was carried out in a commercial cheese packaging plant and involved the insertion of 768 sensors into 384 flow-wrapped cheese packs (two sensors per pack) that were flushed with 100% carbon dioxide prior to sealing. The cheese blocks were randomly assigned to two different storage groups to assess the effects of package quality, packaging process efficiency, and handling and distribution on package containment. Results demonstrated that oxygen levels increased in both experimental groups examined over the 30-day assessment period. The group subjected to a simulated industrial distribution route and handling procedures of commercial retailed cheese exhibited the highest level of oxygen detected on every day examined and experienced the highest rate of package failure. The study concluded that fluctuating storage conditions, product movement associated with distribution activities, and the possible presence of cheese-derived contaminants such as calcium lactate crystals were chief contributors to package failure.

Hypothermia protects brain mitochondrial function from hypoxemia in a murine model of sepsis

Sepsis is commonly associated with brain dysfunction, but the underlying mechanisms remain unclear, although mitochondrial dysfunction and microvascular abnormalities have been implicated. We therefore assessed whether cerebral mitochondrial dysfunction during systemic endotoxemia in mice increased mitochondrial sensitivity to a further bioenergetic insult (hyoxemia), and whether hypothermia could improve outcome. Mice (C57bl/6) were injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg; n = 85) or saline (0.01 ml/g; n = 47). Six, 24 and 48 h later, we used confocal imaging in vivo to assess cerebral mitochondrial redox potential and cortical oxygenation in response to changes in inspired oxygen. The fraction of inspired oxygen (FiO2) at which the cortical redox potential changed was compared between groups. In a subset of animals, spontaneous hypothermia was maintained or controlled hypothermia induced during imaging. Decreasing FiO2 resulted in a more reduced cerebral redox state around veins, but preserved oxidation around arteries. This pattern appeared at a higher FiO2 in LPS-injected animals, suggesting an increased sensitivity of cortical mitochondria to hypoxemia. This increased sensitivity was accompanied by a decrease in cortical oxygenation, but was attenuated by hypothermia. These results suggest that systemic endotoxemia influences cortical oxygenation and mitochondrial function, and that therapeutic hypothermia can be protective.

Cellular ROS imaging with hydro-Cy3 dye is strongly influenced by mitochondrial membrane potential

Background: Hydrocyanines are widely used as fluorogenic probes to monitor reactive oxygen species (ROS) generation in cells. Their brightness, stability to autoxidation and photobleaching, large signal change upon oxidation, pH independence and red/near infrared emission are particularly attractive for imaging ROS in live tissue. Methods: Using confocal fluorescence microscopy we have examined an interference of mitochondrial membrane potential (ΔΨm) with fluorescence intensity and localisation of a commercial hydro-Cy3 probe in respiring and non-respiring colon carcinoma HCT116 cells. Results: We found that the oxidised (fluorescent) form of hydro-Cy3 is highly homologous to the common ΔΨm-sensitive probe JC-1, which accumulates and aggregates only in ‘energised’ negatively charged mitochondrial matrix. Therefore, hydro-Cy3 oxidised by hydroxyl and superoxide radicals tends to accumulate in mitochondrial matrix, but dissipates and loses brightness as soon as ΔΨm is compromised. Experiments with mitochondrial inhibitor oligomycin and uncoupler FCCP, as well as a common ROS producer paraquat demonstrated that signals of the oxidised hydro-Cy3 probe rapidly and strongly decrease upon mitochondrial depolarisation, regardless of the rate of cellular ROS production. Conclusions: While analysing ROS-derived fluorescence of commercial hydrocyanine probes, an accurate control of ΔΨm is required. General significance: If not accounted for, non-specific effect of mitochondrial polarisation state on the behaviour of oxidised hydrocyanines can cause artefacts and data misinterpretation in ROS studies.

Use of fluorescence lifetime imaging microscopy (FLIM) as a timer of cell cycle S phase

Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2’-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.