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.

Quality Characteristics and Shelf-Life of Ultra-High Pressure Homogenized (UHPH) Almond Beverage.

The effects of ultra-high-pressure homogenization (UHPH) at 200 MPa, in combination with different inlet temperatures (55 or 75 °C) during storage at 4 °C were studied and compared with pasteurized (90 °C, 90 s) almond beverage. Microbiological analysis of the physical (particle sedimentation and color) and volatile profile of the most relevant compound in almond beverages was performed at days 1, 7, 14, and 21 of cold storage. UHPH treatment 200 at 75 °C led to higher microbiological reduction after treatment and higher stability during cold storage in almond beverages than pasteurization or UHPH 200 at 55 °C. Physical characteristics of UHPH-treated samples exhibited a high stability during storage with a stable color. Volatile compounds extracted by solid-phase microextraction were identified by gas chromatography coupled with mass spectrometry. The effect of UHPH treatment significantly (p < 0.05) affected the volatile profile compared with pasteurized beverages, although UHPH conditions applied produced similar effects in almond beverages. Benzaldehyde was the most abundant compound detected in all treatments. Hexanal was more abundant in UHPH-treated samples, indicating a higher lipid oxidation compared to pasteurized almond beverages.

Ultra-high-pressure homogenization (UHPH) system for producing high-quality vegetable-based beverages: physicochemical, microbiological, nutritional and toxicological characteristics.

BACKGROUND: A relatively new technology based on a continuous system of ultra-high-pressure homogenization (UHPH) was used for producing high-quality soy and almond beverages as an alternative to conventional heat treatments (pasteurization and UHT). The aim of this study was to compare those treatments by analyzing the most relevant quality parameters with a broad vision from the production to the potential toxicological changes, passing through the main nutritional characteristics. RESULTS: UHPH treatment at 200 MPa, 55 °C T(in) produced a higher reduction of microorganisms than pasteurization. UHPH treatment at 300 MPa, 75 °C T(in) led to complete inactivation of microorganisms, similar to UHT treatment. A much better colloidal stability was observed in both UHPH-treated almond and soy beverages compared with those processed by conventional heat treatments. UHPH treatments led to the same increase in digestibility as heat treatments and did not produce a reduction in the availability of lysine. In addition, UHPH samples of soy beverage seem to be less allergenic based on their lower gut immune response in comparison with heat-treated samples. CONCLUSION: UHPH treatments could be used to produce high-quality commercial vegetable beverages with different quality standards (fresh or long-life storage) according to consumer preference.

Comparing the effects of ultra-high-pressure homogenization and conventional thermal treatments on the microbiological, physical, and chemical quality of almond beverages.

UNLABELLED: The effects of ultra-high-pressure homogenization (UHPH) at 200 and 300 MPa, in combination with different inlet temperatures (55, 65, and 75 °C) on almond beverages with lecithin (AML) and without lecithin (AM), were studied. UHPH-treated samples were compared with the base product (untreated), pasteurized (90 °C, 90 s), and ultra-high-temperature (UHT, 142 °C, 6 s) samples. Microbiological analysis, physical (dispersion stability, particle size distribution, and hydrophobicity), and chemical (hydroperoxide index) parameters of special relevance in almond beverages were studied. Microbiological results showed that pressure and inlet temperature combination had a significant impact on the lethal effect of UHPH treatment. While most UHPH treatments applied produced a higher quality of almond beverage than the pasteurized samples, the combination of 300 MPa and 65 and/or 75 °C corresponded to a maximum temperature after high pressure valve of 127.7 ± 9.7 and 129.3 ± 12.6 °C, respectively. This temperature acted during less than 0.7 s and produced no bacterial growth in almond beverages after incubation at 30 °C for 20 d. UHPH treatments of AML samples caused a significant decrease in particle size, resulting in a high physical stability of products compared to conventional heat treatments. UHPH treatment produced higher values of hydroperoxide index at day 1 of production than heat-treated almond beverage. Hydrophobicity increased in AML-UHPH-treated samples compared to AM and conventional treatments. PRACTICAL APPLICATION: Ultra-high-pressure-homogenization (UHPH) is an emerging technology, a potential alternative to conventional heat treatments. It is a simple process consisting of single step. When liquid food (almond beverage in this study) passes through the high-pressure valve, a very good stability and reduction of microorganisms is achieved, both effects due to the particle breakdown. Specific UHPH conditions could produce commercial sterilization of almond beverage.

Electrochemical detection of Salmonella using gold nanoparticles.

A disposable immunosensor for Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S) detection using a magneto-immunoassay and gold nanoparticles (AuNPs) as label for electrochemical detection is developed. The immunosensor is based on the use of a screen-printed carbon electrode (SPCE) that incorporates a permanent magnet underneath. Salmonella containing samples (i.e. skimmed milk) have been tested by using anti-Salmonella magnetic beads (MBs-pSAb) as capture phase and sandwiching afterwards with AuNPs modified antibodies (sSAb-AuNPs) detected using differential pulse voltammetry (DPV). A detection limit of 143 cells mL(-1) and a linear range from 10(3) to 10(6) cells mL(-1) of Salmonella was obtained, with a coefficient of variation of about 2.4%. Recoveries of the sensor by spiking skimmed milk with different quantities of Salmonella of about 83% and 94% for 1.5×10(3) and 1.5×10(5) cells mL(-1) were obtained, respectively. This AuNPs detection technology combined with magnetic field application reports a limit of detection lower than the conventional commercial method carried out for comparison purposes in skimmed milk samples.

Reduction of counts of Listeria monocytogenes in cheese by means of high hydrostatic pressure.

Inactivation of Listeria monocytogenes (strains NCTC 11994 and Scott A) was evaluated in model cheeses submitted to 10 min HHP treatments of 300, 400 or 500 MPa at 5 or 20 degrees C. Counts were measured immediately after high hydrostatic pressure (HHP) treatment (day 1) and after 2, 15 and 30 days of storage at 8 degrees C. Both strains behaved significantly different after 400 and 500 MPa, being NCTC 11994 more sensitive. Scarce differences were found among final values at both HHP treatment temperatures. Initial reductions (log cfu/g) for 400 MPa at 20 degrees C were 2.9 +/- 0.2 for strain NCTC 11994 and 1.5 +/- 0.2 for Scott A. They reached after 30-day storage 5.3 +/- 0.2 and 4.6 +/- 0.4 log cfu/g for NCTC 11994 and Scott A, respectively. For 500 MPa treatments, day-1 reductions of both strains were around 5-log cfu/g, and counts fell below quantification limit after 30 days. Injured cells (around 0.8-log cfu/g) were mostly observed in 400 MPa treated samples on days 1 and 2. Starter cells suffered higher inactivation and injury. For 20 degrees C treatments, its final counts (log cfu/g) at 300, 400 and 500 MPa were: 8.5 +/- 0.2, 5.4 +/- 0.3 and 2.5 +/- 0.1, respectively. These figures evidence the HHP potential to improve safety of cheese products.

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.

Histidine Decarboxylase Activity of Enterobacter cloacae S15/19 during the Production of Ripened Sausages and Its Influence on the Formation of Cadaverine.

The histidine decarboxylase activity of Enterobacter cloacae S15/19 was studied during the production process of salchichón , a Spanish ripened sausage. Counts of fecal coliform and histidine decarboxylase bacteria decreased during the production process, showing a good correlation in both inoculated and control samples. In the samples inoculated with Enterobacter cloacae S15/19, fecal coliforms were undetectable the last day of the survey, while the population of histidine decarboxylase bacteria was over 2 log MPN/g. Despite the fact that inoculation with Enterobacter cloacae S15/19 increased histidine decarboxylase bacteria counts, no differences were observed in the histamine concentration reached, which was undetectable in most of the control and inoculated samples. In contrast, cadaverine concentration increased significantly (P < 0.01) in the inoculated samples, suggesting that cadaverine could be used as a hygienic-quality indicator of the raw materials employed in sausage processing.

Histidine Decarboxylase Activity of Bacteria Isolated from Raw and Ripened Salchichón , a Spanish Cured Sausage.

Twenty samples of raw sausages (before the ripening process) and 10 of ripened sausages of two different sizes of salchichón , one of the most-consumed ripened meat products in Spain, were microbiologically analyzed. Histidine decarboxylase activity of different isolates obtained from different culture media was evaluated by a decarboxylase agar medium and confirmed afterwards by an enzymatic method. Of the isolates obtained from raw sausages, 15.8% showed histamine-forming activity, most of them (78.1%) belonging to the Enterobacteriaceae family. Klebsiella oxytoca , Enterobacter aerogenes (one isolate), and Enterobacter cloacae (six isolates) showed a powerful histamine-forming activity, producing more than 3,000 µg/ml. In spite of histamine-forming activity being detected in eight of ten analyzed samples of ripened sausages, only one histamine-forming bacterium, identified as Lactobacillus curvatus , was isolated; it showed an ability to form 1,994 µg/ml of histamine in experimental conditions.