Increased inactivation of ozone-treated Clostridium perfringens vegetative cells and spores on fabricated beef surfaces using mild heat

Ozone treatment of beef surfaces enhanced the effectiveness of cooking temperatures ranging from 45 to 75 degrees C against enterotoxin-producing strains of Clostridium perfringens. Vegetative cells on beef surfaces at an initial concentration of 5.59 +/- 0.17 log CFU/g were reduced significantly (P < 0.05) to 4.09 +/- 0.72 log CFU/g and 3.50 +/- 0.90 log CFU/g after combined treatments with aqueous ozone (5 ppm) and subsequent heating at 45 and 55 degrees C, respectively. Spores on the beef surface were likewise significantly reduced from an initial concentration of 2.94 +/- 0.37 log spores per g to 2.07 +/- 0.38 log spores per g and 1.70 +/- 0.37 log spores per g after the combined treatment with aqueous ozone (5 ppm) and subsequent heating at 55 and 75 degrees C, respectively. Fluorescent nucleic acid stains were used with confocal fluorescence microscopy to show that spores remaining attached to the meat were protected from treatment-specific injury. This study provides evidence for the decreased resistance of both vegetative cells and spores of C. perfringens with ozone treatment that is followed by heat treatment at temperatures that would not otherwise be as effective, thus lowering the requirements for cooking beef while maintaining a margin of safety.     

Combined effects of heat, nisin and acidification on the inactivation of Clostridium sporogenes spores in carrot-alginate particles: from kinetics to process validation

Combined effects of mild temperatures, acidification and nisin on the thermal resistance of Clostridium sporogenes ATCC 11437 spores were assessed. Inoculated carrot-alginate particles were used as a solid-food model for the validation of the spore inactivation during the flow of a solid-liquid food system through the holding tube of an aseptic processing unit. Inactivation kinetics was studied in a water bath with the spores inoculated into carrot-alginate particles and in Sorensen's phosphate buffer. For temperatures of 70-90 degrees C, D-values in the buffer were 24.9-5.7 min, much lower than those evaluated for the particles (115.1-22.2 min). Statistical analyses showed significant synergistic effects of temperature and pH on spore inactivation for both media. Acidification reduced the heat resistance of the spores by reducing the D-values. Nisin was not significantly effective at the lower concentrations (up to 750 IU/g). The combination of 90 degrees C, pH: 4.5 and 500IU/g nisin resulted in a ten-fold decrease of the D-value for spores inoculated in the particles (from 111.1 to 10.6 min). Microbial validation tests were conducted using a pilot-scale aseptic processing unit with a mixture of carrot cubes (10%) and carrier liquid of 2%-carboxymethylcellulose solution (90%). Spore-inoculated carrot-alginate particles (initial counts of 106 CFU/g, obtained after come-up-time pre-heat) with pH 3.5 and 2000 IU/g nisin were processed at 90 degrees C in the aseptic processing unit. Microbial analysis showed no spore survivors in the particles after passing through the holding tube (5.2-6.0 min of residence time). The proposed combination of these hurdles significantly enhanced the spore inactivation rate (D(90)=1.17 min) as compared to that for thermal treatment only (D(90)=19.6 min).     

Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll

The aim of this study was to design a thermal treatment(s) for pork luncheon roll, which would destroy Bacillus cereus and Clostridium perfringens vegetative cells and spores. B. cereus and C. perfringens vegetative and spore cocktails were used to inoculate luncheon meat. Samples were subjected to different temperatures and removal times. The decimal-reduction times (D-values) were calculated by linear regression analysis (D = -1/slope of a plot of log surviving cells versus time). The log(10) of the resulting D-values were plotted against their corresponding temperatures to calculate (-1/slope of the curve) the thermal resistance (z-values) of each cocktail. The D-values for vegetative cells ranged from 1 min (60 degrees C) to 33.2 min (50 degrees C) for B. cereus and from 0.9 min (65 degrees C) to 16.3 min (55 degrees C) for C. perfringens. The D-values for B. cereus spores ranged from 2.0 min (95 degrees C) to 32.1 min (85 degrees C) and from 2.2 min (100 degrees C) to 34.2 min (90 degrees C) for C. perfringens. The z-values were calculated to be 6.6 and 8.5 degrees C for B. cereus vegetative and spores, respectively, and 7.8 and 8.4 degrees C for C. perfringens vegetative cells and spores, respectively. The D-values of B. cereus and C. perfringens suggest that a mild cook of 70 degrees C for 12s and 1.3 min would achieve a 6 log reduction of B. cereus and C. perfringens vegetative cells, respectively. The equivalent reduction of B. cereus and C. perfringens spores would require the pork luncheon meat to be heated for 36 s at 105 and 110 degrees C, respectively. The results of this study provide the thermal inactivation data necessary to design a cooking protocol for pork luncheon roll that would inactivate B. cereus and C. perfringens vegetative cells and spores. The data may also be used in future risk assessment studies.     

Modelling the effect of sub(lethal) heat treatment of Bacillus subtilis spores on germination rate and outgrowth to exponentially growing vegetative cells

Spores of Bacillus subtilis were subjected to relatively mild heat treatments in distilled water and properties of these spores were studied. These spores had lost all or part of their dipicolinic acid (DPA) depending on the severity of the heat treatment. Even after relatively mild heat treatments these spore lost already a small but significant amount of DPA. When these spores were inoculated in nutrient medium-tryptone soy broth (TSA)-the non-lethally heated spores started to germinate. Results of classical optical density measurements showed that both phase darkening and subsequent outgrowth could be affected by sub-lethal heat. A study of single cells in TSB showed that lag times originating from exponentially growing cells followed a normal distribution, whereas lag times originating from spores followed a Weibull distribution. Besides classical optical density measurements were made to study the effect of previous heating on the kinetics of the first stages of germination. The germination kinetics could be described by the model as was proposed by Geeraerd et al. [Geeraerd, A.H., Herremans, C.H. and Van Impe, J.F., 2000. Structural model requirements to describe microbial inactivation during a mild heat treatment. International Journal of Food Microbiology 59, 185-209]. Two of the 4 parameters of the sigmoid model of Geeraerd were dependent on heating time and heating temperature, whereas the two other parameters were considered as independent of the heating conditions. Based on these observations, a secondary model could be developed that describes the combined effect of heating temperature and heating time on the kinetics of germination. To have more detailed information of the kinetics of germination samples incubated in TSB were tested at regular time intervals by flow cytometry. To that end the cells were stained with syto 9 to distinguish between the various germination stages. There was a qualitative agreement between the results of flow cytometry and those of optical density measurements, but there was a difference in quantitative terms. The results have shown that germination rate of spores is dependent on previous heating conditions both in the first stage when phase darkening occurs and also during the later stages of outgrowth when the phase dark spore develops to the vegetative cell.     

Short communication: lack of breed differences in responses of bovine spermatozoa to heat shock

An experiment was conducted to test whether the magnitude of effects of heat shock on spermatozoal function were less for thermotolerant breeds (Brahman and other breeds with Brahman influence) than for breeds that evolved in northern Europe (Angus and Holstein). Frozen spermatozoa were thawed, purified by Percoll gradient centrifugation and incubated at 38.5, 41, or 42 degrees C for 4 h. Sperm motility was then analyzed with a Hamilton Thorn Motility Analyzer. Heat shock reduced the percentage of sperm that were motile, mean track speed, and mean path velocity. There were no significant breed x temperature interactions for these traits. The mean frequency of tail beat tended to be reduced by heat shock in bulls of Brahman-influenced breeds and, to a lesser extent, in Brahman bulls, but it was not affected by heat shock in Angus or Holstein bulls. For no traits were there significant temperature x bull within breed interactions. Overall, results indicate that 1) heat shock reduces motility of bovine spermatozoa and 2) genetic effects are unlikely to be an important determinant of the function of ejaculated sperm following heat shock.     

Genetic divergence in cellular resistance to heat shock in cattle: differences between breeds developed in temperate versus hot climates in responses of preimplantation embryos,reproductive tract tissues and lymphocytes to increased culture temperatures

The detrimental effects of heat stress on fertility in cattle are less pronounced in heat-tolerant breeds. Although these genetic differences reflect differences in thermoregulation, cells from heat-tolerant breeds are less adversely compromised by increased temperature (that is, heat shock) than cells from heat-sensitive breeds. Experiments were performed to test the hypothesis that cells and tissues from two thermotolerant breeds (Brahman and Senepol) are better able to survive and function after exposure to increased temperature than cells and tissues from two thermosensitive breeds (Holstein and Angus). Exposure of embryos at>eight-cell stage at day 5 after insemination to heat shock of 41.0 degrees C for 6 h decreased development to the blastocyst stage and the number of cells per embryo. However, the deleterious effect of heat shock on blastocyst formation and the number of cells per embryo was less pronounced for Brahman than for Holstein and Angus breeds. Embryos from Senepol cows had very low development and it was not possible to determine heat shock effects in this breed. In contrast to the sensitivity of embryos to heat shock, there was no effect of a 41.0 degrees C heat shock on [(3)H]leucine incorporation into proteins secreted by oviductal or endometrial explants. Lymphocytes from Brahman and Senepol cows were more resistant to heat-induced apoptosis than lymphocytes from other breeds. Heat shock reduced lymphocyte glutathione content but the magnitude of the decrease was not affected by breed. In conclusion, embryos from Brahman cows are more resistant to heat shock than embryos from Holstein or Angus cows. Genetic differences are also present in thermotolerance for apoptosis response in lymphocytes, with Brahman and Senepol cattle being more resistant to heat shock than Angus and Holstein breeds. It is likely that the evolutionary forces that led to the Brahman and Senepol breeds being adapted to hot climates resulted in the selection of genes controlling resistance to cellular heat shock.     

Modifications to methods for the enumeration and detection of injured Escherichia coli O157:H7 in foods

Reliable methods are required for the detection and enumeration of potentially injured E. coli O157 in food in the presence of outnumbering competing bacteria. Selective agents can prevent or inhibit the recovery and subsequent multiplication of injured cells and direct inoculation, either into selective enrichment broths or onto selective agar plates is still used in many methods for E. coli O157 detection and enumeration. When compared with tryptone soya agar (TSA), sorbitol MacConkey agar (SMAC) was shown to underestimate the concentration of viable E. coli O157:H7 subjected to low pH and high NaCl concentration. Using a resuscitation stage on TSA followed by membrane transfer to SMAC improved recovery to levels obtained on TSA. The membrane method was used to monitor the numbers of artificially contaminated E. coli O157:H7 during the fermentation of a meat product and demonstrated better survival when compared to counts on SMAC. Six rapid methods for the detection of E. coli O157 in food (BAX E. coli O157, Reveal 8 E. coli O157-H7 screening test, VIP EHEC, VIDAS E. coli O157 (ECO), EHEC-Tek and Tecra E. coli O157 visual immunoassay), were evaluated using beetburgers, parsley and fermented meat artificially contaminated with injured cells. Methods using direct selective enrichment, with or without an elevated incubation temperature gave false-negative results. The incorporation of a non-selective pre-enrichment medium improved the detection rates of these assays by up to ten fold.     

Visual expression analysis of the responses of the alternative oxidase gene (aox1) to heat shock, oxidative, and osmotic stresses in conidia of citric acid-producing Aspergillus niger

The citric acid-producing filamentous fungus Aspergillus niger WU-2223L shows cyanide-insensitive respiration catalyzed by alternative oxidase in addition to the cytochrome pathway. Sequence analysis of the 5' flanking region of the alternative oxidase gene (aox1) revealed a potential heat shock element (HSE) and a stress response element (STRE). We have previously confirmed aox1 expression in conidia. In this study, to confirm whether the upstream region of aox1 responds to various stresses, we used a visual expression analysis system for single-cell conidia of the A. niger strain AOXEGFP-1. This strain harbored a fusion gene comprising aox1 and egfp, which encodes the enhanced green fluorescent protein (EGFP). The fluorescence intensity of EGFP increased in conidia of A. niger AOXEGFP-1 that were subjected to heat shock at 35-45 °C, oxidative stress by exposure to 5mM paraquat or 1 mM t-butylhydroperoxide, or osmotic stresses by exposure to 0.5 M KCl or 1.0 M mannitol. These results indicate that the putative HSE and STRE in the upstream region of aox1 directly or indirectly respond to heat shock, oxidative, and osmotic stresses.     

Efficacy of heat treatment in the reduction of salmonellae and Escherichia coli O157:H– on alfalfa, mung bean and radish seeds used for sprout production

We studied the effect of hot-water treatment at various time/temperature regimes to design a decontamination process which is consistent with the recommendation of the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) to reduce pathogens on seeds by 5log cfu/g. Alfalfa, mung bean and radish seeds were inoculated by immersion with more than 10⁷ cfu/g of enterobacteria (Salmonella Senftenberg W775, S. Bovismorbificans and Escherichia coli O157:H^–), dried and stored at 2 °C. The numbers of salmonellae and E. coli O157:H^– on these seeds remained unchanged during storage for 8 weeks. To achieve sprouting rates of more than 95%, time-temperature regimes were defined. The thermal treatment of contaminated mung bean (2–20 min for 55–80 °C), radish and alfalfa seeds 0.5–8 min (53–64 °C) reduced all pathogens by more than 5log cfu/g. For S. Senftenberg W775 on radish seeds, D values of 3.2, 1.9 and 0.6 min were determined for exposure at 53, 55 and 58 °C and a z value of 6.2 °C was calculated. For alfalfa seeds, the respective D values were 3.0, 1.6, and 0.4 min and the z value was the same as that determined for radish seeds.     

Use of global sensitivity analysis in quantitative microbial risk assessment: application to the evaluation of a biological time temperature integrator as a quality and safety indicator for cold smoked salmon

The aim of this study was to apply a global sensitivity analysis (SA) method in model simplification and to evaluate (eO)^®, a biological Time Temperature Integrator (TTI) as a quality and safety indicator for cold smoked salmon (CSS). Models were thus developed to predict the evolutions of Listeria monocytogenes and the indigenous food flora in CSS and to predict TTIs endpoint. A global SA was then applied on the three models to identify the less important factors and simplify the models accordingly. Results showed that the subset of the most important factors of the three models was mainly composed of the durations and temperatures of two chill chain links, out of the control of the manufacturers: the domestic refrigerator and the retail/cabinet links. Then, the simplified versions of the three models were run with 10⁴ time temperature profiles representing the variability associated to the microbial behavior, to the TTIs evolution and to the French chill chain characteristics. The results were used to assess the distributions of the microbial contaminations obtained at the TTI endpoint and at the end of the simulated profiles and proved that, in the case of poor storage conditions, the TTI use could reduce the number of unacceptable foods by 50%.