Mark T. Morgan

Detection of Low Levels of Listeria monocytogenes Cells by Using a Fiber-Optic Immunosensor

ABSTRACT Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 × 103 CFU/ml for a pure culture of L. monocytogenes grown at 37°C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10°C with 3.5% NaCl, the detection threshold was 4.1 × 104 or 2.8 × 107 CFU/ml, respectively. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth.

Development of technology for the early post mortem prediction of water holding capacity and drip loss in fresh pork.

Two different technologies were tested on the slaughterline for their ability to predict drip loss at 24 h, namely near infrared reflectance (NIR) and impedance measurements using a tetra polar measuring geometry at a frequency of 1000 Hz. The results demonstrate that NIR measurements (900-1800 nm) acquired during a 6 min period starting only 30 min post exsanguination through a fibre optic probe in combination with multivariate data analysis can be used for predicting drip loss 24 h after slaughter. A correlation higher than 0.8 was observed for a trial on 99 carcasses measured at a commercial slaughterhouse. The tetrapolar impedance measurements did not perform as well as NIR yielding a correlation of 0.5 with 24 h drip loss.

Use of high-concentration-short-time chlorine dioxide gas treatments for the inactivation of Salmonella enterica spp. inoculated onto Roma tomatoes

Salmonella outbreaks have been recently linked to the consumption of fresh tomatoes. Thus, there is a need to develop systems that reduce the risk of microbial contamination to increase product shelf-life and keep fresh fruit attributes. The objectives of this study were to evaluate high-concentration-short-time chlorine dioxide gas treatments effects on Salmonella-inoculated Roma tomatoes and determine the optimal treatment conditions for microbial inactivation and shelf-life extension. Effects of ClO(2) concentration (2, 5, 8 and 10mg/l) and exposure time (10, 30, 60, 120 and 180 s) on inoculated Roma tomatoes were studied. Salmonella enterica strains, serotype Montevideo, Javiana and Baildon, were used to experimentally inoculate the food product. After ClO(2) treatments, tomatoes were stored at room temperature for 28 days. Inherent microbial population, change in tomato color, and chlorine dioxide gas residuals were evaluated. ANOVA analysis showed that both ClO(2) concentration and treatment time were significant (p<0.01) for Salmonella inactivation. Surviving Salmonella populations of 3.09, 2.17 and 1.16 logCFU/cm(2) were obtained treating tomatoes with 8 mg/l ClO(2) for 60 s, 10 mg/l ClO(2) for 120 s, and 10 mg/l for 180 s, respectively (initial Salmonella population: 6.03±0.11 log CFU/cm(2)). The selected treatments significantly reduced background microflora (p<0.05), while fruit color and residual contents were not significantly different (p>0.05), as compared to the control. Results suggest the potential for high-concentration-short-time treatments ClO(2) gas as an effective pathogen inactivation technology for large-scale produce packing operations.

Evanescent Wave Fiber Optic Biosensor for Salmonella Detection in Food

Salmonella enterica is a major food-borne pathogen of world-wide concern. Sensitive and rapid detection methods to assess product safety before retail distribution are highly desirable. Since Salmonella is most commonly associated with poultry products, an evanescent wave fiber-optic assay was developed to detect Salmonella in shell egg and chicken breast and data were compared with a time-resolved fluorescence (TRF) assay. Anti-Salmonella polyclonal antibody was immobilized onto the surface of an optical fiber using biotin-avidin interactions to capture Salmonella. Alexa Fluor 647-conjugated antibody (MAb 2F-11) was used as the reporter. Detection occurred when an evanescent wave from a laser (635 nm) excited the Alexa Fluor and the fluorescence was measured by a laser-spectrofluorometer at 710 nm. The biosensor was specific for Salmonella and the limit of detection was established to be 103 cfu/mL in pure culture and 104 cfu/mL with egg and chicken breast samples when spiked with 102 cfu/mL after 2–6 h of enrichment. The results indicate that the performance of the fiber-optic sensor is comparable to TRF, and can be completed in less than 8 h, providing an alternative to the current detection methods.

Effects of microwave on the drying, checking and mechanical strength of baked biscuits

Abstract The influence of high-frequency radiation on quality in terms of checking and mechanical strength of low-moisture baked products was investigated. Biscuits baked in a convection, reel oven were compared to biscuits of very similar moisture content and appearance that were baked in the same oven until the falling-rate drying period but then immediately microwaved for 30 s in a 700 W microwave oven. Microwave baking was found to significantly reduce checking to 5% compared to 61% in conventional biscuits. The average breaking stress required for intact microwaved biscuits two weeks after baking was 997 kPa, while that for conventional samples was 610 kPa. The structure of conventional biscuits was generally found to weaken at a faster rate than microwaved biscuits. Microwaved biscuits were also found to be less susceptible to checking upon exposure to high ambient humidity. In addition, the finite element software, FIDAP, was used to predict heat and mass transfer with respect to time and position during microwaving under various conditions. A biscuit was modeled as an axisymmetric short cylinder. Simulations confirmed greater drying rates and more uniform internal moisture profiles for microwaved biscuits. A minimum microwaving time of approximately 28 s was predicted to be necessary to produce a uniform moisture profile for a power absorption of 1.7 W/g. An elevation in the biscuit temperature during baking as well as a more uniform moisture distribution found in microwaved biscuits were thought to be critical factors in determining checking behavior and mechanical strength.

Evaluation of chlorine dioxide gas residues on selected food produce.

In recent years, the consumption of fresh fruits and vegetables has greatly increased, and so has its association with contamination of several foodborne pathogens (Listeria, Salmonella, and Escherichia coli). Hence, there is a need to investigate effective sanitizer systems for produce decontamination. Chlorine dioxide (ClO(2)), a strong oxidizing gas with broad spectrum and sanitizing properties, has previously been studied for use on selected fruits and vegetables. ClO(2) gas treatments show great potential for surface pathogen reduction; however its use from a residue safety standpoint has yet to be assessed. Thus, the objective of this study was to evaluate residues of ClO(2), chlorite, chlorate, and chloride on selected fresh produce surfaces after treatment with ClO(2) gas. A rinse procedure was used and water samples were analyzed by N, N-diethyl-p-phenylenediamine and ion chromatography method (300.0). Seven different foods--tomatoes, oranges, apples, strawberries, lettuce, alfalfa sprouts, and cantaloupe--were analyzed after ClO(2) treatment for surface residues. Very low residues were detectable for all the food products except lettuce and alfalfa sprouts, where the measured concentrations were significantly higher. Chlorine dioxide technology leaves minimal to no detectable chemical residues in several food products, thus result in no significant risks to consumers. Practical Application: Potential for chlorine dioxide gas treatments as an effective pathogen inactivation technology to produce with minimal risk for consumers.

A comparative study on the effectiveness of chlorine dioxide gas, ozone gas and e-beam irradiation treatments for inactivation of pathogens inoculated onto tomato, cantaloupe and lettuce seeds

The increase in reported food-borne outbreaks linked with consumption of raw fruits and vegetables has motivated new research focusing on prevention of pre-harvest produce contamination. This study evaluates and compares the effectiveness of three non-thermal technologies, chlorine dioxide gas, ozone gas and e-beam irradiation, for inactivation of Salmonella enterica and Escherichia coli O157:H7 on pre-inoculated tomato, lettuce and cantaloupe seeds, and also their corresponding effect on seeds germination percentage after treatments. Samples were treated with 10mg/l ClO(2) gas for 3 min at 75% relative humidity, with 4.3mg/l ozone gas for 5 min and with a dose of 7 kGy electron beam for 1 min. Initial load of pathogenic bacteria on seeds was ~6 log CFU/g. Results demonstrate that all treatments significantly reduce the initial load of pathogenic bacteria on seeds (p<0.05). In particular, after ozone gas treatments 4 log CFU/g reduction was always observed, despite the seeds and/or microorganisms treated. ClO(2) and e-beam treatments were noticeably more effective against Salmonella on contaminated tomato seeds, where 5.3 and 4.4 log CFU/g reduction were respectively observed. Germination percentage was not affected, except for cantaloupe seeds, where the ratio was significantly lowered after ClO(2) treatments. Overall, the results obtained show the great applicability of these non-thermal inactivation techniques to control and reduce pathogenic bacteria contamination of seeds.

Detection of Salmonella enteritidis Using a Miniature Optical Surface Plasmon Resonance Biosensor

The frequent outbreaks of foodborne illness demand rapid detection of foodborne pathogens. Unfortunately, conventional methods for pathogen detection and identification are labor-intensive and take days to complete. Biosensors have shown great potential for the rapid detection of foodborne pathogens. Surface plasmon resonance (SPR) sensors have been widely adapted as an analysis tool for the study of various biological binding reactions. SPR biosensors could detect antibody-antigen bindings on the sensor surface by measuring either a resonance angle or refractive index value. In this study, the feasibility of a miniature SPR sensor (Spreeta, TI, USA) for detection of Salmonella enteritidis has been evaluated. Anti-Salmonella antibodies were immobilized on the gold sensor surface by using neutravidin. Salmonella could be detected by the Spreeta biosensor at concentrations down to 105 cfu/ml.

The application of high-concentration short-time chlorine dioxide treatment for selected specialty crops including Roma tomatoes (Lycopersicon esculentum), cantaloupes (Cucumis melo ssp. melo var. cantaloupensis) and strawberries (Fragaria × ananassa)

The effects of high-concentration short-time chlorine dioxide (ClO2) gas treatment on food-borne pathogens inoculated onto the surface of tomatoes, cantaloupes, and strawberries were studied. Produce were spot-inoculated with a mixture of Salmonella enterica (serotypes Montevideo, Javiana and Baildon), Escherichia coli O157:H7 (serotypes 204 P, EDL 933 and C792) or Listeria monocytogenes (serotypes Scott A, F 5069 and LCDC 81-861), and treated with ClO2 gas at 10 mg/l for 180 s. After ClO2 gas treatment, surviving populations were determined and shelf-life studies were conducted (microbial spoilage population, change in color and overall appearance). Significant microbial reduction (p < 0.05) was observed for all treated samples. Nearly a 5LogCFU/cm(2)Salmonella reduction was found on tomatoes, cantaloupe and strawberries, while a ~3LogCFU/cm(2) reduction was observed for E. coli and Listeria on all produce surfaces. E. coli and Listeria appeared to be more resistant to ClO2 gas as compared to Salmonella spp. Treatments significantly (p < 0.05) reduced initial microflora population, while produce color surface was not significantly influenced, as compared to the control (p > 0.05). Results obtained suggest the potential use of high-concentration short-time ClO2 gas treatment as an effective online pathogen inactivation technology for specialty crops in large-scale produce packing operations.

Optical immunosensors for detection of Listeria monocytogenes and Salmonella enteritidis from food

Listeria monocytogenes and Salmonella are two major foodborne pathogens of significant concern. Two optical evanescent wave immunosensors were evaluated for detection: Antibody-coupled fiber-optic biosensor and a surface plasmon resonant (SPR) immunosensor. In the fiber-optic sensor, polyclonal antibodies for the test organisms were immobilized on polystyrene fiber wave -guides using streptavidin - biotin chemistry. Cyanine 5 -labeled monoclonal antibodies C11E9 (for L. monocytogenes) and SF-11 (for Salmonella Enteritidis) were used to generate a specific fluorescent signal. Signal acquisition was performed by launching a laser-light (635 nm) from an Analyte-2000. This immunosensor was able to detect 103 - 109 cfu/ml of L. monocytogenes or 106-109 cfu/ml of Salmonella Enteritidis and the assays were conducted at near real-time with results obtained within one hour of sampling. The assays were specific and showed signal even in the presence of other microorganisms such as E. coli, Enterococcus faecalis or Salmonella Typhimurium. In the SPR system, IAsys instrument (resonant mirror sensor) was used. Monoclonal antibody-C11E9 was directly immobilized onto a carboxylate cuvette. Whole Listeria cells at various concentrations did not yield any signal while surface protein extracts did. Crude protein extracts from L. monocytogenes and L. innocua had average binding responses of around 150 arc sec (0.25 ng/mm2), which was significantly different from L. grayi, L. ivanovii, or L. welshimeri with average responses of <48 arc sec. Both fiber-optic and SPR sensors show promise in near real-time detection of foodborne L. monocytogenes and Salmonella Enteritidis.