A Diffusion Model to Quantify Membrane Repair Process in Listeria monocytogenes Exposed to High Pressure Processing Based on Fluorescence Microscopy Data.

The effects of environmental stresses on microorganisms have been well-studied, and cellular responses to stresses such as heat, cold, acids, and salts have been extensively discussed. Although high pressure processing (HPP) is becoming more popular as a preservation method in the food industry, the characteristics of the cellular damage caused by high pressure are unclear, and the microbial response to this stress has not yet been well-explored. We exposed the pathogen Listeria monocytogenes to HPP (400 MPa, 8 min, 8°C) and found that the high pressure created plasma membrane pores. Using a common staining technique involving propidium iodide (PI) combined with high-frequency fluorescence microscopy, we monitored the rate of diffusion of PI molecules into hundreds of bacterial cells through these pores on days 0, 1, 2, 3, and 4 after pressurization. We also developed a mathematical dynamic model based on mass transfer and passive diffusion laws, calibrated using our microscopy experiments, to evaluate the response of bacteria to HPP. We found that the rate of diffusion of PI into the cells decreased over the 4 consecutive days after exposure to HPP, indicating repair of the pressure-created membrane pores. The model suggested a temporal change in the size of pores until closure. To the best of our knowledge, this is the first time that pressure-created membrane pores have been quantitatively described and shown to diminish with time. In addition, we found that the membrane repair rate in response to HPP was linear, and growth was temporarily arrested at the population level during the repair period. These results support the existence of a progressive repair process in some of the cells that take up PI, which can therefore be considered as being sub-lethally injured rather than dead. Hence, we showed that a subgroup of bacteria survived HPP and actively repaired their membrane pores.

From hazard analysis to risk control using rapid methods in microbiology: A practical approach for the food industry.

The prevention of foodborne diseases is one of the main objectives of health authorities. To this effect, analytical techniques to detect and/or quantify the microbiological contamination of foods prior to their release onto the market are required. Management and control of foodborne pathogens have generally been based on conventional detection methodologies, which are not only time-consuming and labor-intensive but also involve high consumable materials costs. However, this management perspective has changed over time given that the food industry requires efficient analytical methods that obtain rapid results. This review covers the historical context of traditional methods and their passage in time through to the latest developments in rapid methods and their implementation in the food sector. Improvements and limitations in the detection of the most relevant pathogens are discussed from a perspective applicable to the current situation in the food industry. Considering efforts that are being done and recent developments, rapid and accurate methods already used in the food industry will be also affordable and portable and offer connectivity in near future, which improves decision-making and safety throughout the food chain.

Influence of final baking technologies in partially baked frozen gluten-free bread quality.

The effect of final baking in convection oven (FBC), microwave oven (FBM), and microwave oven with susceptor packaging material (FBMS) on partially baked (PB) frozen gluten-free bread characteristics was investigated. Specific volume and crust color of loaves were measured at day 0. Bread moisture, water activity, and crumb and crust texture (at 15, 45, and 90 min after baking) were analyzed at day 0 and after 28 d of frozen storage (-18 °C). Volatile compounds from breads baked in convection oven or microwave oven with susceptor packaging material were also evaluated. Bread finally baked in convection oven or in microwave oven with susceptor packaging increased crust browning. Crumb and roll hardness increased with time after final baking (measured at 15, 45, 90 min) and after 28 d of frozen storage. Bread finally baked in microwave oven was the hardest, due to high water losses. At day 0, bread finally baked in convection oven had softer crumb than bread finally baked in microwave oven with susceptor packaging but, after 28 d of frozen storage, there were no differences between them. Moreover, FBC and FBMS rendered gluten-free breads that could not be distinguished in a triangular test and had the same volatile compounds profile. In conclusion, FBMS could be an alternative to FBC.

Effect of tiger nut-derived products in gluten-free batter and bread.

Tiger nut is a tuber used to produce tiger nut milk that yields a high quantity of solid waste, which can be dried and used as fiber source. The objective of this paper was to evaluate the quality of gluten-free bread formulated with different tiger nut-derived products in order to substitute soya flour (which is an allergen ingredient) and, at the same time, increase the use of tiger nut-derived products. Four gluten-free formulations based on corn starch and containing tiger nut milk, tiger nut milk by-product, tiger nut flour, or soya flour (as reference formulation) were studied. Tiger nut milk increased G' of gluten-free batter and rendered breads with the softest crumb (502.46 g ± 102.05), the highest loaf-specific volume (3.35 cm(3)/g ± 0.25), and it was mostly preferred by consumers (61.02%). Breads elaborated with tiger nut flour had similar characteristics than soya flour breads (except in color and crumb structure). The addition of tiger nut milk by-product resulted in a hard (1047.64 g ± 145.74) and dark (L(*) = 70.02 ± 3.38) crumb bread, which was the least preferred by consumers. Results showed that tiger nut is a promising ingredient to formulate gluten-free baked products.

Flow cytometry immunodetection and membrane integrity assessment of Escherichia coli O157:H7 in ready-to-eat pasta salad during refrigerated storage.

Over the past years, products of non-animal origin have been increasingly linked to foodborne diseases caused by the enterohemorrhagic pathogen Escherichia coli O157:H7. Contaminated fresh produce and derived ready-to-eat meals are of major concern, since no further or only minimal processing is applied. In this study, flow cytometry was evaluated as a rapid technique to detect E. coli O157:H7 by immunofluorescence, using polyclonal antibodies conjugated to R-phycoerythrin, in refrigerated ready-to-eat pasta salad containing acetic acid and benzoic acid. Signal filtering strategies were applied during sample analysis to reduce the limit of detection of the technique to 5 log CFU/g. Simultaneously with pathogen detection, physiological state was assessed by staining with the membrane integrity indicators propidium iodide and SYBR Green I. Fine tuning of dye concentrations and ratios allowed discrimination of not only cells with intact or damaged membranes, but also of cells with partially damaged membranes, which were considered injured cells. Then, changes in membrane integrity of inoculated E. coli O157:H7 cells were monitored throughout 14-day refrigerated storage. Most cells were injured at the beginning of refrigeration, but showed an intact membrane at the end. This suggests that injured E. coli O157:H7 cells underwent a membrane repair during exposure to refrigeration and acid stresses, and survived in ready-to-eat pasta salad. This highlights the importance of the implementation of control measures to limit the presence of this pathogen in non-animal origin food products. Additionally, the proposed immunodetection and membrane integrity three-color assay in food is a good tool to monitor the effect of a number of food-related treatments on E. coli O157:H7 cell membrane.

Fate of Escherichia coli strains inoculated in model cheese elaborated with or without starter and treated by high hydrostatic pressure.

The aim of this research was to study high hydrostatic pressure inactivation of two strains of Escherichia coli (E. coli O59:H21 [CECT 405] and E. coli O157:H7 [CECT 5947]) inoculated in washed-curd model cheese elaborated with and without starter and the ability of these strains for survival, recovery, and growth. Samples were treated at 300, 400, and 500 MPa for 10 min at 20 degrees C and analyzed after the treatment and after 1, 7, and 15 days of storage at 8 degrees C to study the behavior of Escherichia populations. Cheeses elaborated with starter showed the maximum lethality at 400 and 500 MPa, and no significant differences in the baroresistant behavior of either strains were detected, except for E. coli O157:H7 at 400 MPa in cell counts obtained with thin agar layer method medium, where the decrease value was significantly lower. In cheese elaborated without starter, the highest decrease value was observed at 500 MPa, except for E. coli O59:H21 in cell counts obtained with selective culture medium, where the highest decrease value was also found at 400 MPa. The ability to repair and grow was not observed in model cheese elaborated with starter, as cell counts of treated samples decreased after 15 days of storage at 8 degrees C. By contrast, in cheese elaborated without starter, all pressurized samples showed the trend to repair and grow during the storage period in both strains. These results suggest that the presence of starter and low pH values are the main factors that control the ability of Escherichia strains inoculated in this type of cheese and treated by high hydrostatic pressure to recover and grow.

Survival and growth of Yersinia enterocolitica strains inoculated in skimmed milk treated with high hydrostatic pressure.

Four human pathogenic strains of Yersinia enterocolitica (serotypes O:1, O:3, O:8, and O:9) were inoculated (7-8 log CFU/ml) in UHT skimmed milk and treated at 300, 400, and 500 MPa for 10 min at 20 degrees C, and then kept at 8 degrees C to assess their evolution for 15 days. Treatments at 400 and 500 MPa caused the highest lethality, generally reaching counts below detection level (1 CFU/ml) in the culture media. At 300 MPa, the most baroresistant serotypes were O:3 and O:8. After 15 days of storage at 8 degrees C, Y. enterocolitica showed growth over 8 log (CFU/ml) in all treatments. Kinetic study of microbial inactivation in skimmed milk was performed with serotype O:8 at 300 MPa, showing a tailing after 35 min of pressure treatment.

Behavior of Yersinia enterocolitica strains inoculated in model cheese treated with high hydrostatic pressure.

The effects of high hydrostatic pressure treatment and the ability for survival, repair, and growth of three human pathogenic serotypes (O:1, O:3, O:8) of Yersinia enterocolitica were investigated in washed-curd model cheese made with pasteurized bovine milk. Samples were treated at 300, 400, and 500 MPa for 10 min at 20 degrees C and analyzed at 0, 1, 7, and 15 days to assess the viability of the Yersinia population. A long-term study (up to 60 days of ripening after high hydrostatic pressure treatment) was also undertaken. Treatments at 400 and 500 MPa caused maximum lethality, and only the treatment at 300 MPa showed significant differences (P < 0.05) between serotypes; the most baroresistant was O:3. Ability to repair and grow was not observed after 15 days of storage at 8 degrees C. Yersinia counts in untreated cheese samples also decreased below the detection limit at day 45 in the long-term study. These results suggest that the cheese environment did not allow recovery of injured cells or growth. A primary contributing factor to this effect seemed to be the low pH resulting from the production of lactic acid during cheese ripening.

Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme.

The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme. The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese. Cheeses were vacuum packed and stored at 8 degrees C. All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments. Bacillus cereus counts were measured 24 h and 15 d after HHP treatment. The combination of both cycles improved the efficiency of the whole treatment. When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP. For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment. Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.

Populations of Aerobic Mesophils and Inoculated E. coli during Storage of Fresh Goat's Milk Cheese Treated with High Pressure.

Pasteurized goat's milk inoculated with Escherichia coli 405 CECT was manufactured into cheese containing 108CFU/g. The fresh cheese was treated by combinations of pressure (400, 450, and 500 MPa), temperature (2, 10, and 25°C) and time (5, 10, and 15 min). Once treated, cheeses were stored at 2 to 4°C. Counts of surviving Escherichia coli and aerobic mesophilic bacteria were determined 1, 15, 30, and 60 days after treatment. No colonies of surviving E. coli were detected 1 day after pressurization, except in samples treated for 5 min at 25°C at pressures of 400 and 450 MPa. No surviving E. coli were detected at 15, 30, or 60 days in any case. Aerobic mesophilic bacteria counts after treatment were between 2 and 3 log CFU/g in most cases and only a slight increase during refrigerated storage could be detected in samples treated at 400 MPa.