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.     

Persistence strategies of Bacillus cereus spores isolated from dairy silo tanks

Survival of Bacillus cereus spores of dairy silo tank origin was investigated under conditions simulating those in operational dairy silos. Twenty-three strains were selected to represent all B. cereus isolates (n = 457) with genotypes (RAPD-PCR) that frequently colonised the silo tanks of at least two of the sampled eight dairies. The spores were studied for survival when immersed in liquids used for cleaning-in-place (1.0% sodium hydroxide at pH 13.1, 75 °C; 0.9% nitric acid at pH 0.8, 65 °C), for adhesion onto nonliving surfaces at 4 °C and for germination and biofilm formation in milk. Four groups with different strategies for survival were identified. First, high survival (log 15 min kill ≤1.5) in the hot-alkaline wash liquid. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterised by slow germination in rich medium and well preserved viability when exposed to heating at 90 °C. Fourth, spores capable of germinating at 8 °C and possessing the cspA gene. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot-alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus.     

Growth and inhibition by spices of growth from spores of enterotoxigenic Bacillus cereus in cooked rice

Bacillus cereus is a pathogenic spore-forming bacterium implicated in cases of diarrheal and emetic type of foodborne illness. We previously found that enterotoxigenic B. cereus is widely present in retail spices. Here we analyzed the spore heat resistance of nine diarrheal strains isolated from spices. Seven had D_95°C values ranging from 0.64 to 3.53 min while two emetic strains had D_95°C values of 7.04 min and 6.64 min. The ability of selected strains to grow in cooked rice at temperatures 20 °C, 17 °C and 12 °C was determined as well as their toxin expression capability. After 48 h, B. cereus levels of 1.26 × 10⁷ and 3.8 × 10⁷ CFU/g were obtained in cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 10⁴ CFU/g even after 48 h of incubation. The increase in the aerobic, mesophilic bacterial population (APC) and B. cereus population naturally present in 0.1% crushed pepper added to cooked rice was determined over a period of 48 h at 20 °C and 17 °C. Levels of B. cereus in pepper/rice samples reached a maximum of 1600 MPN/g at 20 °C while the APC was 2.4 × 10⁸/g at this temperature. When thyme, containing the same initial natural level of B. cereus, was added to rice in place of pepper, more than a five-fold greater level of B. cereus was detected at 20 °C. Since thyme contained initial APC of 2.5 log₁₀ less than pepper we conclude that the high APC functions in a competitive-exclusion manner, minimizing the growth of B. cereus and potentially other agents of foodborne illness. This is particularly relevant in instances of temperature abuse of foods and may explain the low incidence of foodborne illness due to B. cereus despite its widespread presence shown in previous surveys of market spices.     

Mathematical modelling of Aspergillus ochraceus inactivation with supercritical carbon dioxide – A kinetic study

The aim of this research was to analyze and model the combined effect of pressure and temperature upon Aspergillus ochraceus spores exposed to high pressure carbon dioxide (HPCD) treatment and to estimate the kinetic parameters. Lately, many empirical or semi-empirical mathematical models were presented and discussed for different microorganisms, mainly bacteria, demonstrating an increased need for tools able to quantify the parameters of the microbial inactivation. A. ocharaceus HPCD inactivation was adequately described by first order reaction kinetics and a synergic effect of pressure and temperature was noticed for the experimental range where pressure varied from 5.4 to 7.0 MPa and temperature varied from 30 to 50 °C. The decimal reduction time (D) ranged from 47.07 min at 5.4 MPa and 30 °C to 5.04 min at 7.0 MPa and 50 °C. In this study three mathematical models were evaluated in order to find the best one that describes accurately the influence of pressure and temperature on the studied microbial response. An empirical exponential equation, that described pressure and temperature influence in the form of a polynomial equation, was found to best describe the dependence of A. ochraceus HPCD inactivation in the range 5.4–7.0 MPa and 30–50 °C.This work adds insight to moulds inactivation at the already existing body of knowledge on bacteria inactivation with HPCD and provides support to potential industrial applications of the minimal–thermal methods that combine high pressure and mild temperature with carbon dioxide for different food matrixes.     

Bacillus probiotics

Bacterial spore formers are being used as probiotic supplements for use in animal feeds, for human dietary supplements as well as in registered medicines. Their heat stability and ability to survive the gastric barrier makes them attractive as food additives and this use is now being taken forward. While often considered soil organisms this conception is misplaced and Bacilli should be considered as gut commensals. This review summarises the current use of Bacillus species as probiotics, their safety, mode of action as well as their commercial applications.     

The fate of Clostridium perfringens spores exposed to ozone and/or mild heat pretreatment on beef surfaces followed by modified atmosphere packaging

Clostridium perfringens is a natural contaminant of raw beef products that can proliferate to dangerous cell levels under conditions of temperature abuse. Spores of the bacterium were inoculated onto irradiated London broil beef at levels of 3 log₁₀ spores/g beef. Samples of beef (7.5×10.0×1.0 cm) were treated with aqueous ozone (5 ppm O₃ for 5 min), or heat (60°C for 30 min), or both and then vacuum-packaged to 2 kPa for up to 10 d storage at 37°C, 25°C, or 4°C. Storage at 37°C resulted in increases in viable counts after 1 d to over 7 log₁₀ cfu/g beef, whereas storage at 4°C prevented spore germination and growth for all treatments. At 25°C, heat-treated beef samples reached 6 log₁₀ cfu/g viable counts in 2 d and spores/vegetative cells on control or ozone-treated samples did not germinate or grow through the first day of vacuum-packaged storage. Modified atmospheres with increasing CO₂ concentration were also compared with regard to bacterial survival during beef storage at 25°C. C. perfringens spores remained dormant in control and ozone-treated beef during a 10-d storage at 25°C. Pretreatment with heat increased germination and outgrowth during storage of beef, whereas ozone treatment and no treatment controls were effective in inhibiting spore germination and outgrowth in combination with increasing CO₂ concentrations above 30% or refrigeration. These data support the avoidance of heat in the pretreatment of raw beef.     

The control of Clostridium botulinum during extended storage of pressure-treated,cooked chicken

High pressure processing (HPP) is used as a post-process decontamination treatment to ensure that cooked chicken is free from Listeria monocytogenes and other food poisoning bacteria. However HPP does not inactivate bacterial endospores and the conditions in cooked chicken could support the growth of Clostridium botulinum. Therefore some method for controlling the germination of spores and their subsequent growth would be necessary to ensure a safe product over an extended storage period.A five strain cocktail of non-proteolytic C. botulinum was inoculated into raw chicken mince, which was cooked and then pressure treated at 600 MPa for 2 min at 20 °C. C. botulinum survived cooking and HPP and subsequently grew in the pressure-treated, cooked chicken during storage. Germination and growth was controlled when 2% w/w sodium lactate was added and when oxygen-permeable packaging was used. Weissella viridescens was not suitable for use as a protective culture to control the growth of C. botulinum.     

Impacts of water quality on chlorine and chlorine dioxide efficacy in natural waters

The impact of disinfection efficacy in natural waters was evaluated by performing disinfection assays using four untreated surface waters of various qualities and ultra-pure buffered waters as a baseline condition for comparison. Bacillus subtilis spores were spiked in these waters and disinfection assays were conducted at 22 degrees C using either free chlorine or chlorine dioxide. Assays using indigenous aerobic spores were also completed. The inactivation kinetics in natural and ultra-pure buffered waters were not statistically different (at p = 0.05) while using free chlorine, as long as disinfectant decay was taken into account. Filtering natural waters through a 0.45 microm did not improve the sporicidal efficacy of chlorine. For three out of the four waters tested, the efficacy of chlorine dioxide was greater in natural waters compared to that observed in ultra-pure buffered waters. Such results are consistent with previous observations using ultra-pure waters supplemented with NOM-extract from the Suwannee River. Similar to free chlorine results, the impact of filtration (0.45 microm) on the efficacy of chlorine dioxide was not statistically significant.     

Investigation of Bacillus cereus contamination sites in a dairy plant with RAPD-PCR

The usefulness of RAPD-PCR as a typing method for investigation of Bacillus cereus isolates from a dairy plant was evaluated. A 1-d in-depth sampling study along the entire production line was performed and long-term sampling of products was conducted over two years. In addition, farm bulk tank isolates were collected. Isolates from an individual sample during storage at 7°C were often identical. Large clusters of identical isolates were found in the silo tank in the 1-d study and in samples from the long-term study. In combination with analytical data, these results indicated a prolonged contamination problem caused by a mesophilic strain early in the production chain. Detailed and useful information on occurrence of psychrotrophic B. cereus was obtained along the production line and among products. RAPD-PCR appeared to be a useful typing method for B. cereus isolates. In combination with carefully planned sampling and microbiological analysis, RAPD could be helpful in solving dairy contamination problems in pasteurized milk.     

Distributions of the growth rate of the germ tubes and germination time of Penicillium chrysogenum conidia depend on water activity

The effects of water activities for sporulation (a(wsp)) and germination (a(wge)) on the distributions of the growth rate of the germ tubes (mu) and the germination time (t(G)) of Penicillium chrysogenum conidia were determined by monitoring the length of the same germ tubes throughout the experiments automatically. No relationship between the individual t(G)'s and mu's could be established. Irrespective of the water activity for germination, mu was greater and t(G) was less for conidia produced at 0.95a(wsp) than that at 0.99a(wsp). At 0.99 a(wge) the mean and the standard deviation of t(G) were smaller than those obtained at 0.95a(wge). At 0.99a(wge), normal distributions for mu and t(G) were exhibited, but not at 0.95a(wge). The cumulative frequencies were used to reconstruct the germination curves. Great differences in the percentage of spores capable of germination (P(G)) and in the mean germination times between conidia produced at 0.95a(wsp) and at 0.99a(wsp) were clearly exhibited at 0.95a(wge), thus demonstrating the paramount influence of sporulation conditions on germination kinetics.