Effects of DNA extraction methods on the real time PCR quantification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in chicken feces and ceca contents.

Polymerase chain reaction (PCR) method was coupled with a DNA extraction to enumerate Campylobacter spp. from poultry gastrointestinal tract samples. Three experiments were conducted that included: 1) Development of a DNA standard curve related to bacterial DNA primers; 2) Design of a cell/genomic DNA extraction protocol to isolate Campylobacter spp. DNA from complex samples such as poultry feces; and 3) Comparison of PCR quantification to standard plate count methodology. The standard curve using primers for Campylobacter spp. was created for DNA extracted from environmental isolates with a linear range (R2 > 0.95) and with a high specificity for C. coli and C. jejuni recovered from poultry, swine and laboratory isolates. A 2-step extraction process of bacterial DNA from poultry feces was developed in which the cells were first concentrated using a gradient-centrifugation step followed by comparison of 4 DNA extraction methods. Two commercial DNA extraction methods (Zymo Research Quick DNA, and Invitrogen magnetic separation), a traditional phenol-chloroform DNA extraction method using proteinase K to inactivate DNAses, and an in-house isolation method for DNA extraction based on chaotropic salts were used. The middle gradient layer recovered 89% to 98% of the bacteria cells from the sample, with recovery dependent upon the Campylobacter genus. The 4 DNA extractions methods recovered 112 to 302 ug/nL of DNA. Finally, the qPCR and standard plate methods were highly correlated for enumerating Campylobacter spp. in the 2.0 to 8.0-log CFU range. Analyses of the results from this study demonstrate that the combination of the standard curve for Campylobacter spp. DNA primers, the gradient cell concentration method and DNA extraction techniques with qPCR can be used to enumerate Campylobacter spp. from poultry samples with findings similar those of traditional plate count methodology.

The Use of Organic Rankine Cycles for Recovering the Heat Lost in the Compression Area of a Cryogenic Air Separation Unit.

The use of organic Rankine cycles (ORCs) is a viable solution for the recovery of waste heat. For an air separation unit (ASU) with a production of V˙O2=58300mN3/h operating in Romania, the value of utilization of the heat transferred to the cooling system of the compression area represents 21% of the global system electrical energy input. To recover this thermal energy and transform it into mechanical energy, an ORC system was proposed. To maximize the production of mechanical power, an exergy analysis was performed. Exergy analysis was used to choose the most suitable organic fluid and find the optimum constructive structure of the Rankine cycle. The calculation of the exergy destruction in the key apparatuses of the system allowed investigation into the optimization search procedure. The large exergy destruction in the liquid preheater suggested the decrease in the temperature difference in this part of the evaporator by increasing the inlet temperature of the liquid; and an internal recuperative heat exchanger was used for this purpose. When permitted, the overheating of the vapors also reduced the temperature difference between the heat source and the organic fluid during the heat transfer process. The analysis was comparatively performed for several organic fluids such as R-245fa, R123, n-pentane and R717. The use of ammonia, that offered the possibility of superheating the vapors at the turbine inlet, brought a gain of mechanical power corresponding to 6% economy in the electrical energy input of the global plant.

Exergetic Analysis of a Cryogenic Air Separation Unit.

This case study analyzes a cryogenic air separation unit (ASU) with a production of V˙O2=58,300 [m3Nh] of gaseous oxygen with a concentration greater than 98.5%, operating in Romania on a steel plant platform. The goal of the paper is to provide an extensive model of exergetic analysis that could be used in an optimization procedure when decisional parameters are changed or structural design modifications are implemented. For each key part of the Air Separation Unit, an exergetic product and fuel were defined and, based on their definition, the coefficient of performance of each functional zone was calculated. The information about the magnitude of the exergetic losses offers solutions for their future recovery. The analysis of the exergy destructions suggests when it is worth making a larger investment. The exergetic analysis of the compression area of the ASU points out an exergy destruction and loss of 37% from the total plant's electrical energy input. The exergy loss with the heat transferred to the cooling system of compressors can be recovered; for the exergy destruction portion, the challenge between investment and operating costs should be considered. The exergy destruction of the air separation columns found the High Pressure Column (HPC) to be more destructive than the Low Pressure Column. The share of the exergy destruction in the total plant's electrical energy input is 8.3% for the HPC. The local COP of the HPC, calculated depending on the total exergy of the local product and fuel, is 62.66%. The calculus of the air separation column is performed with the ChemSep simulator.

Inhibition of Listeria monocytogenes in Hot Dogs by Surface Application of Freeze-Dried Bacteriocin-Containing Powders from Lactic Acid Bacteria.

Six lactic acid bacteria (LAB) strains, Lactococcus lactis BFE 920, L. lactis subsp. lactis ATCC 11454, L. lactis subsp. cremoris ATCC 14365, Lactobacillus curvatus L442, Lact. curvatus LTH 1174, and Lact. bavaricus MN, were grown in cheddar cheese whey supplemented with complex nutrient sources. Cell-free culture supernatants were freeze-dried, and the resulting bacteriocin-containing powders were applied on the surface of hot dogs that were inoculated (~4 log cfu/hot dog) with a five-strain Listeria monocytogenes cocktail. Hot dogs were vacuum-sealed and stored at 4 °C for 4 weeks. L. monocytogenes was enumerated, using both tryptic soy agar (TSA) and oxford listeria agar (OXA), on day 0 and at 1, 2, 3, and 4 weeks of the refrigerated storage. In hot dogs containing only the L. monocytogenes inoculum, L. monocytogenes counts increased from 4 up to 7 log cfu/hot dog. All samples containing freeze-dried bacteriocin-containing powders exhibited significantly lowered (P < 0.05) L. monocytogenes populations on the surface of hot dogs throughout the 4-week study except for bavaricin MN powder. Bacterial counts on hot dogs packed without any powder were statistically equal on day 0 when enumerated on OXA. Freeze-dried bacteriocin-containing powders from Lact. curvatus L442 and L. lactis subsp. cremoris ATCC 14365 decreased L. monocytogenes populations on the surface of hot dogs by greater than 2 log cfu/hot dog throughout the 4-week study. For the powdered bacteriocin preparations from L. lactis BFE 920, L. lactis subsp. lactis ATCC 11454, and Lact. curvatus LTH 1174, L. monocytogenes populations were determined to be approximately 3-log cfu/hot dog after 4 weeks of storage.

Production of Antilisterial Bacteriocins from Lactic Acid Bacteria in Dairy-Based Media: A Comparative Study.

One hundred and eight strains of lactic acid bacteria (LAB) were screened for bacteriocin production by the modified deferred antagonism and agar well diffusion methods. When the modified deferred antagonism method was employed, 82 LAB strains showed inhibitory action against Listeria monocytogenes v7 ½a, whereas 26 LAB strains expressed no inhibition. Only 12 LAB strains exhibited inhibitory activity when the agar well diffusion method was used, 11 of which had been previously recognized as bacteriocin production positive (Bac(+)). Lactobacillus viridescens NRRL B-1951 was determined, for the first time, to produce an inhibitory compound with a proteinaceous nature. The inhibitory activity was observed in the presence of lipase, α-chymotrypsin, and trypsin, but no inhibition zone could be detected in the presence of proteinase K, indicating the proteinaceous nature of the inhibitory compound. The inhibitory compound was active against Lact. sake ATCC 15521 and Lact. plantarum NCDO 995. Bacteriocin production by the Bac(+) LAB strains was assessed in Lactobacillus MRS Broth as well as in dairy-based media such as nonfat milk, demineralized whey powder, and cheddar cheese whey supplemented with complex nutrient sources that are rich in nitrogen. Lact. sake ATCC 15521 and L. monocytogenes CWD 1002, CWD 1092, CWD 1157, CWD 1198, and v7 ½a were used as indicators. The inhibitory activities of the bacteriocins varied depending on the indicator strains and the growth media used. The LAB indicator strains were found to be more sensitive to inhibition by bacteriocins when compared to the listerial indicator strains. Among the listerial indicators, L. monocytogenes CWD 1002 and CWD 1198 were the most sensitive strains to the bacteriocins investigated in this study. Media composition had a significant influence on bacteriocin production and activity. When compared to demineralized whey powder medium and cheddar cheese whey medium supplemented with whey protein concentrate, cheddar cheese whey medium supplemented with complex nutrient sources such as yeast extract, polypeptone, proteose peptone nr. 3, or soytone appeared to be more supportive of bacteriocin production.

Effects of Chicken Litter Storage Time and Ammonia Content on Thermal Resistance of Desiccation-Adapted Salmonella spp.

Broiler chicken litter was kept as a stacked heap on a poultry farm, and samples were collected up to 9 months of storage. Chicken litter inoculated with desiccation-adapted Salmonella cells was heat-treated at 75, 80, 85, and 150°C. Salmonella populations decreased in all these samples during heat treatment, and the inactivation rates became lower in chicken litter when storage time was extended from 0 to 6 months. There was no significant difference (P > 0.05) in thermal resistance of Salmonella in 6- and 9-month litter samples, indicating that a threshold for thermal resistance was reached after 6 months. Overall, the thermal resistance of Salmonella in chicken litter was affected by the storage time of the litter. The changes in some chemical, physical, and microbiological properties during storage could possibly contribute to this difference. Moisture and ammonia could be two of the most significant factors influencing the thermal resistance of Salmonella cells in chicken litter. Our results emphasize the importance of adjusting time and temperature conditions for heat processing chicken litter when it is removed from the chicken house at different time intervals.

Thermal inactivation of desiccation-adapted Salmonella spp. in aged chicken litter.

Thermal inactivation of desiccation-adapted Salmonella spp. in aged chicken litter was investigated in comparison with that in a nonadapted control to examine potential cross-tolerance of desiccation-adapted cells to heat treatment. A mixture of four Salmonella serovars was inoculated into the finished compost with 20, 30, 40, and 50% moisture contents for a 24-h desiccation adaptation. Afterwards, the compost with desiccation-adapted cells was inoculated into the aged chicken litter with the same moisture content for heat treatments at 70, 75, 80, 85, and 150°C. Recovery media were used to allow heat-injured cells to resuscitate. A 5-log reduction in the number of the desiccation-adapted cells in aged chicken litter with a 20% moisture content required >6, >6, ∼4 to 5, and ∼3 to 4 h of exposure at 70, 75, 80, and 85°C, respectively. As a comparison, a 5-log reduction in the number of nonadapted control cells in the same chicken litter was achieved within ∼1.5 to 2, ∼1 to 1.5, ∼0.5 to 1, and <0.5 h at 70, 75, 80, and 85°C, respectively. The exposure time required to obtain a 5-log reduction in the number of desiccation-adapted cells gradually became shorter as temperature and moisture content were increased. At 150°C, desiccation-adapted Salmonella cells survived for 50 min in chicken litter with a 20% moisture content, whereas control cells were detectable by enrichment for only 10 min. Our results demonstrated that the thermal resistance of Salmonella in aged chicken litter was increased significantly when the cells were adapted to desiccation. This study also validated the effectiveness of thermal processing being used for producing chicken litter free of Salmonella contamination.