Heat sensitization of hepatitis A virus and Tulane virus using grape seed extract, gingerol and curcumin.

Human noroviruses (HNoV) and hepatitis A virus (HAV) are predominantly linked to foodborne outbreaks worldwide. As cell-culture systems to propagate HNoV in laboratories are not easily available, Tulane virus (TV) is used as a cultivable HNoV surrogate to determine inactivation. Heat-sensitization of HAV and TV by "generally recognized as safe'' (GRAS) substances can potentially reduce their time-temperature inactivation parameters during processing to ensure food safety. Curcumin, gingerol (from ginger), and grape seed extract (GSE) reportedly have anti-inflammatory, immune-modulating and antiviral properties. The objective of this study was to determine and compare the D-values and z-values of HAV and TV at 52-68 °C with or without curcumin (0.015 mg/ml), gingerol (0.1 mg/ml), or GSE (1 mg/ml) in 2-ml glass vials. HAV at ~7 log PFU/ml and TV at ~6 log PFU/ml were diluted in phosphate buffered saline (PBS) and added to two sets of six 2-mL sterile glass vials. One set served as the control and the second set had the three extracts individually added for thermal treatments in a circulating water bath for 0-10 min. The D-values for TV in PBS ranged from 4.55 ± 0.28 to 1.08 ± 0.16 min, and for HAV in PBS ranged from to 9.21 ± 0.24 to 0.67 ± 0.19 min at 52-68 °C. Decreased D-values (52-58 °C) for TV with curcumin ranging from 4.32 ± 0.25 to 0.62 ± 0.17 min, gingerol from 4.09 ± 0.18 to 0.72 ± 0.09 min and GSE from 3.82 ± 0.18 to 0.80 ± 0.07 min, with similar trends for HAV were observed. The linear model showed significant differences (p < 0.05) between the D-values of HAV and TV with and without plant extracts for most tested temperatures. This suggests that GRAS substances can potentially lower temperature and time regimens needed to inactivate HAV and TV.

Transcriptional and phenotypic responses of Listeria monocytogenes to chlorine dioxide.

Significant food-borne disease outbreaks have occurred from consumption of ready-to-eat foods, including produce, contaminated with Listeria monocytogenes. Challenging food matrices (e.g., cantaloupe, sprouts) with limited processing steps postharvest to reduce pathogen loads have underscored a need for new mitigation strategies. Chlorine dioxide (ClO2) is increasingly being used in produce and other food systems to reduce food-borne pathogen levels. The goal of this study was to characterize the transcriptional response and survival of L. monocytogenes 10403S exposed to ClO2. The transcriptional profile of log-phase cells exposed to 300 mg/liter ClO2 for 15 min was defined by whole-genome microarray. A total of 340 genes were significantly differentially expressed. Among the differentially expressed genes, 223 were upregulated (fold change ≥ 1.5; adjusted P value < 0.05) in role categories responsible for protein fate, cellular processes, and energy metabolism. There were 113 and 16 genes differentially expressed belonging to regulatory networks of σ(B) and CtsR, respectively. We assessed L. monocytogenes 10403S survival after exposure to 100, 300, and 500 mg/liter aqueous ClO2 in brain heart infusion (BHI) broth; there was a significant difference between cells exposed to 500 mg/liter ClO2 and those exposed to all other conditions over time (P value < 0.05). Isogenic ΔsigB and ΔctsR mutants exposed to 300 mg/liter ClO2 were more sensitive to ClO2 than the wild type under the same conditions. These results provide an initial insight into the mechanisms that L. monocytogenes employs to survive sublethal ClO2 and further our understanding of the inactivation mechanisms of this increasingly used sanitizer.

Use of chlorine dioxide gas for the postharvest control of Alternaria alternata and Stemphylium vesicarium on Roma tomatoes.

BACKGROUND: Tomatoes and potatoes are the top produce affected in terms of value lost in the USA. Postharvest losses can occur anywhere from the time of harvest to the consumers' decision to eat or discard the food. These data support the importance of finding sustainable strategies to minimise food waste and preserve resources. This study evaluated the potential application of chlorine dioxide gas (ClO2 ) technology to control the postharvest spoilage of Roma tomatoes by Alternaria alternata and Stemphylium vesicarium. RESULTS: Data analysis showed that exposure time was a significant factor for fungal disease control (P < 0.05). After 3 min of treatment, mycelial growth was completely inhibited for A. alternata and S. vesicarium. Similar results were observed for conidial germination. The efficacy of ClO2 treatments was also studied under in vivo conditions. While untreated Roma tomatoes developed white moulds and black spots after 5 days of storage, produce decay was significantly (P < 0.05) delayed after 5 and 7 min treatments for S. vesicarium and A. alternata respectively. CONCLUSION: The use of ClO2 in the food industry is regulated by both the FDA and the EPA. Currently, only acidified sodium chlorite solutions are approved for the control of micro-organisms in water used to wash fruits and vegetables. No direct applications of ClO2 gas on fresh fruits and vegetables can be found in the regulations. More data are required by the two agencies to demonstrate that residues of ClO2 on produce surfaces are acceptable for human consumption.

Development of online closed-loop frequency shifting strategies to improve heating performance of foods in a solid-state microwave system.

The solid-state microwave generator is promising to replace magnetron as a power source in domestic ovens for its precise and flexible control over a wide range of operational parameters and its potential to improve heating performance. Shifting frequency during microwave heating, either orderly or using complementary heating patterns, has yielded better heating performance than traditional single-frequency heating. This study developed three online frequency shifting strategies (orderly, pre-determined complementary, and dynamic complementary) that simultaneously collected heating performances and provided closed-loop feedback through customized algorithms to control the frequency shifting during the microwave heating processes. Each algorithm was implemented and tested on two model foods with different dielectric properties (gellan gel and mashed potato). The three frequency shifting algorithms had similar frequency sweeping processes but considerably different frequency shifting procedures for different replications and food products. The dynamic complementary frequency shifting strategy simultaneously evaluated the heating performance and determined the next-step complementary frequency. The method had shown better heating uniformity than the orderly and pre-determined complementary frequency shifting strategies. The dynamic complementary frequency shifting strategy could accommodate different food products and can be incorporated into future smart microwave ovens.

Aichi virus inactivation by heat in 2-ml glass vials.

Aichi virus (AiV) that results in gastroenteritis worldwide, is spread through contaminated shellfish and water. The resistance/tolerance of AiV to common inactivation processes along with the absence of commercially available vaccines makes it necessary to study its thermal inactivation kinetics. This research evaluated the heat inactivation of AiV in cell-culture media using 2-ml sterile glass vials by the linear and Weibull models. Heat treatments of AiV titers of 7 log plaque forming units (PFU)/ml were conducted thrice in a water-bath at 50, 54, and 58 °C for up to 90 min. Plaque assays for each dilution in duplicate were used to determine infectious virus titers. Linear model D-values for AiV at 50 ± 1 °C (± = standard error) (come-up time = 68 s), 54 ± 0.7 °C (130 s), and 58 ± 0.6°C (251 s) were 43.3 ± 4.23 (R2 = 0.40, RMSE = 0.56), 5.69 ± 0.28 (R2 = 0.80, RMSE = 0.43), and 1.20 ± 0.63 min (R2 = 0.69, RMSE = 0.39), respectively, and the linear model z-value was 5.14 ± 0.39°C (R2 = 0.99, RMSE = 0.08). For the same temperatures, the Weibull model td = 1 values were 20.98 ± 8.8 (R2 = 0.62, RMSE = 0.46, α (scale parameter) = 2.30, ß (shape parameter) = 0.38), 3.84 ± 0.69 (R2 = 0.85, RMSE = 0.38, α = 1.08, ß = 0.66), and 0.87 ± 0.10 min (R2 = 0.80, RMSE = 0.32, α = 0.22, ß = 0.61), respectively and the z-value (using Td = 1 ) was 5.79 ± 0.22 °C (R2 = 1.0, RMSE = 0.03). A better fit was obtained with the Weibull model for log reductions versus time with higher R2 and lower RMSE values. Application of AiV inactivation parameters can help reduce the risk of AiV outbreaks.

Comparison of inactivation of Listeria monocytogenes within a biofilm matrix using chlorine dioxide gas, aqueous chlorine dioxide and sodium hypochlorite treatments.

The present research compared the effect of chlorine dioxide (CD) gas, aqueous CD and aqueous sodium hypochlorite (SHC) treatments on the inactivation of a five strain mixture of Listeria monocytogenes - containing biofilms. Four day old biofilms were developed on a stainless steel (SS 304) coupon by using a mixture of five cultures of L. monocytogenes (Scott A, N1-227, 103M, 82 and 311) using a 100% relative humidity (RH) dessicator for incubation at room temperature (22 ± 2 °C). After biofilm development, coupons were rinsed and dried for 2 h and treated with 0.3 mg/l CD gas at 75% RH, 7 mg/l of aqueous CD and 50 mg/l SHC. Initial log(10) population of biofilm cells before CD gas, aqueous CD and SHC treatment was 4.80, 5.09 and 4.95 log(10) CFU/cm(2). The Weibull model was used to fit non-linear survivor curves. Treatments and time points of 0.3 mg/l CD gas and 7 mg/l aq. CD solution were significantly different (p < 0.05). A 10 min treatment of 0.3 mg/l CD gas, 7 mg/l of aq. CD, and 50 mg/l SHC resulted in reductions of 3.21, 3.74 and 3.09 log(10) CFU/cm(2), respectively. At 10 min, all treatments were not statistically different (p > 0.05). Low levels of CD (0.3 mg/l CD gas and 7 mg/l aq. CD solution) for 10 min resulted in similar log reductions compared to 50 mg/l SHC.

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 10(3) cfu/mL in pure culture and 10(4) cfu/mL with egg and chicken breast samples when spiked with 10(2) 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.

Thermal Inactivation Kinetics of Tulane Virus in Cell-Culture Medium and Spinach.

Human noroviruses (HNoVs) cause significant gastrointestinal disease outbreaks worldwide. Tulane virus (TV) is a cultivable HNoV surrogate widely used to determine control measures against HNoVs. The objective of this study was to determine the heat inactivation kinetics (D- and z-values) of TV in cell-culture media and on spiked homogenized spinach using the first-order and Weibull models. TV in cell-culture media at approximately 7 log PFU/mL (PFU-plaque forming unit) in 2-mL glass vials was heated at 52, 54, and 56 °C for up to 10 min in a circulating water bath. Survivors were enumerated using confluent host LLC-MK2 cells in six-well plates by plaque assay. Data from three replicate treatments assayed in duplicate were analyzed statistically. D-values by the first-order model for TV in cell-culture media at 52, 54, and 56 °C were 4.59 ± 0.05, 2.91 ± 0.05, and 1.74 ± 0.07 min, respectively, with a z-value of 9.09 ± 0.01 °C (R2  = 0.997). The Weibull model showed td  = 1 values of 2.53 ± 0.08, 1.99 ± 0.10, and 0.57 ± 0.64 min, respectively, at the same temperatures. The D-values for TV in spinach were 7.94 ± 0.21, 4.09 ± 0.04, and 1.43 ± 0.02 min and the z-value was 10.74 ± 0.01 °C (R2  = 0.98) by the first-order model and 4.89 ± 0.02, 3.21 ± 0.45, and 0.25 ± 0.38 min for the Weibull model at 50, 54, and 58 °C, respectively. In comparison to previously reported results for the cultivable HNoV surrogate, murine norovirus -1, TV in cell-culture media and spiked on spinach homogenates showed lower D- and z-values. TV may not be an ideal HNoV surrogate for heat inactivation studies in cell-culture media or homogenized spinach in vacuum bags.

Essential Oils Against Pathogen and Spoilage Microorganisms of Fruit Juices: Use of Versatile Antimicrobial Delivery Systems.

Essential oils (EO) are increasingly used as natural antimicrobial compounds, however the effect of delivery system to enhance their antimicrobial activity has not been widely studied. Limonene (0 to 10 µL/mL) was added to microbial suspensions (∼105 CFU/mL) of selected foodborne pathogens (Listeria monocytogenes Scott A, Salmonella enterica Typhimurium, Escherichia coli and Staphylococcus aureus), and spoilage microorganisms (Lactobacillus plantarum, Saccharomyces cerevisiae, and Candida albicans). S. aureus was found to be the most sensitive foodborne pathogen while Salmonella enterica showed continued growth under all concentrations. Stable nanoemulsions and solid lipid nanoparticles (SLN) (d ∼ 170 nm) were prepared using an alkane carrier oil (n-tetradecane and n-eicosane, respectively). Interfacial effects and homogenous distribution of limonene in nanoemulsions improved its (8 and 12 µL/mL) antimicrobial effect against S. aureus. Higher aqueous concentrations as a result of expulsion from SLN further enhanced the antimicrobial activity pronounced at higher limonene concentrations. Therefore, our findings confirm that the emulsion-based delivery systems are able to effectively distribute limonene inside a microbial suspension to improve its antimicrobial activity.

Adhesion characteristics of Listeria adhesion protein (LAP)-expressing Escherichia coli to Caco-2 cells and of recombinant LAP to eukaryotic receptor Hsp60 as examined in a surface plasmon resonance sensor.

Listeria adhesion protein (LAP) is an important adhesion factor in Listeria monocytogenes and interacts with its cognate receptor, mammalian heat shock protein 60 (Hsp60). The genetic identity of LAP was determined to be alcohol acetaldehyde dehydrogenase (Aad). A recombinant Escherichia coli strain expressing aad confirmed the involvement of Aad in adhesion to Caco-2 cells. Binding kinetics (ka) of recombinant LAP (rLAP) to Hsp60 was examined in a surface plasmon resonance sensor and was determined to be 5.35 x 10(8) M(-1) s(-1) and it was equivalent to the binding of anti-Hsp60 antibody (ka = 2.15 x 10(9) M(-1) s(-1)) to Hsp60. In contrast, Internalin B, an adhesion/invasion protein from L. monocytogenes, used as a control, had binding kinetics (ka) of only 2.9 x 10(6) M(-1) s(-1). The KD value of rLAP was 1.68 x 10(-8) M, which was significantly lower than Internalin B (KD = 6.5 x 10(-4) M). These results suggest that Hsp60 has significantly higher avidity for anti-Hsp60 antibody and LAP than Internalin B. In summary, LAP is identified as an alcohol acetaldehyde dehydrogenase and binding of recombinant E. coli to Caco-2 cells or rLAP to Hsp60 protein was found to be highly specific.

The effect of a novel low temperature-short time (LTST) process to extend the shelf-life of fluid milk.

Pasteurization has long been the standard method to extend the shelf-life of dairy products, as well as a means to reduce microbial load and the risk of food-borne pathogens. However, the process has limitations, which include cost effectiveness, high energy input, and reduction of product quality/organoleptic characteristics. In an effort to reduce these limitations and extend shelf-life, this study examined a novel low temperature, short time (LTST) method in which dispersed milk in the form of droplets was treated with low heat/pressure variation over a short treatment time, in conjunction with pasteurization. Lactobacillus fermentum and Pseudomonas fluorescens Migula were exposed to conventional pasteurization treatments with and without LTST. Using these organisms, the LTST addition was able to reduce microbial load below detection limits; 1.0 × 10(1) cfu/mL, from approximately 1.2 × 10(8) and 1.0 × 10(7) cfu/mL for L. fermentum and P. fluorescens Migula, respectively. In addition, the shelf-life of the treated, raw, and uninoculated product was prolonged from 14 to 35 days, compared with standard pasteurization, to as long as 63 days with the LTST amendment. Sensory analysis of samples also demonstrated equal or greater preference for LTST + pasteurization treated milk when compared to pasteurization alone (α = 0.05). Conventional pasteurization was effective at reducing the above mentioned microorganisms by as much as 5.0 log10 cfu/mL. However, LTST was able to achieve 7.0-8.0 log10 cfu/mL reduction of the same microorganisms. In addition, BActerial Rapid Detection using Optical scattering Technology detected and identified microorganisms isolated both pre- and post-treatment, of which the only organisms surviving LTST were Bacillus spp. Increased lethality, improved shelf-life, and equal or better organoleptic characteristics without increased energy consumption demonstrate the effectiveness of the incorporation of LTST. The improved shelf-life may potentially have major impacts in the dairy industry in terms of shipping and overall sustainability.

Chloroxyanion Residue Quantification in Cantaloupes Treated with Chlorine Dioxide Gas.

Previous studies show that treatment of cantaloupes with chlorine dioxide (ClO2) gas at 5 mg/liter for 10 min results in a significant reduction (P < 0.05) in initial microflora, an increase in shelf life without any alteration in color, and a 4.6- and 4.3-log reduction of Escherichia coli O157:H7 and Listeria monocytogenes, respectively. However, this treatment could result in the presence of chloroxyanion residues, such as chloride (Cl(-)), chlorite (ClO2(-)), chlorate (ClO3(-)), and perchlorate (ClO4(-)), which, apart from chloride, are a toxicity concern. Radiolabeled chlorine dioxide ((36)ClO2) gas was used to describe the identity and distribution of chloroxyanion residues in or on cantaloupe subsequent to fumigation with ClO2 gas at a mean concentration of 5.1 ± 0.7 mg/liter for 10 min. Each treated cantaloupe was separated into rind, flesh, and mixed (rind and flesh) sections, which were blended and centrifuged to give the corresponding sera fractions. Radioactivity detected, ratio of radioactivity to mass of chlorite in initial ClO2 gas generation reaction, and distribution of chloroxyanions in serum samples were used to calculate residue concentrations in flesh, rind, and mixed samples. Anions detected on the cantaloupe were Cl(-) (∼ 90%) and ClO3(-) (∼ 10%), located primarily in the rind (19.3 ± 8.0 µg of Cl(-)/g of rind and 4.8 ± 2.3 µg of ClO3(-)/g of rind, n = 6). Cantaloupe flesh (∼ 200 g) directly exposed to(36)ClO2 gas treatment showed the presence of only Cl(-) residues (8.1 ± 1.0 µg of Cl(-)/g of flesh, n = 3). Results indicate chloroxyanion residues Cl(-) and ClO3(-) are only present on the rind of whole cantaloupes treated with ClO2 gas. However during cutting, residues may be transferred to the fruit flesh. Because Cl(-) is not toxic, only ClO3(-) would be a toxicity concern, but the levels transferred from rind to flesh are very low. In the case of fruit flesh directly exposed to ClO2 gas, only nontoxic Cl(-) was detected. This indicates that ClO2 gas that comes into contact with edible flesh would not pose a health concern.

Hybridoma Ped-2E9 cells cultured under modified conditions can sensitively detect Listeria monocytogenes and Bacillus cereus.

Lymphocyte origin hybridoma Ped-2E9 cell-based cytotoxicity assay can detect virulent Listeria or Bacillus species, and its application in a cell-based biosensor for onsite use would be very attractive. However, maintaining enough viable cells on a sensor platform for a prolonged duration is a challenging task. In this study, key factors affecting the survival and growth of Ped-2E9 cells under modified conditions were investigated. When the Ped-2E9 cells were grown in media containing 5% fetal bovine serum in sealed tubes without any replenishment of nutrients or exogenous CO(2) supply, a large portion of the cells remained viable for 6 to 7 days and cells entered into G0/G1 resting phase. The media pH change was negligible and no cell death was observed in the first 4 days, then cells sequentially underwent apoptotic (fourth day onward) phase until day 7 after which a majority was dead. Subsequent cytotoxicity testing of 3- to 7-day stored Ped-2E9 cells sensitively detected virulent Listeria and Bacillus species. These data strongly suggest that Ped-2E9 cells can be maintained in viable state for 6 days in a sealed tube mimicking the environment in a potential sensor device for onsite use without the need for expensive cell culture facilities.

Detection of low levels of Listeria monocytogenes cells by using a fiber-optic immunosensor.

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 x 10(3) CFU/ml for a pure culture of L. monocytogenes grown at 37 degrees C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10 degrees C with 3.5% NaCl, the detection threshold was 4.1 x 10(4) or 2.8 x 10(7) 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.