Juan S. Aguirre

Analysis of the Variability in the Number of Viable Bacteria after Mild Heat Treatment of Food

ABSTRACT Variability in the numbers of bacteria remaining in saline solution and whole milk following mild heat treatment has been studied with Listeria innocua, Enterococcus faecalis, Salmonella enterica serovar Enteritidis, and Pseudomonas fluorescens. As expected, the most heat-resistant bacterium was E. faecalis, while P. fluorescens was the least heat resistant, and all bacteria showed greater thermal resistance in whole milk than in saline solution. Despite the differences in the inactivation kinetics of these bacteria in different media, the variability in the final number of bacteria was affected neither by the species nor by the heating substrate, but it did depend on the intensity of the heat treatment. The more severe the heat treatment was, the lower the average number of surviving bacteria but the greater the variability. Our results indicated that the inactivation times for the cells within a population are not identically distributed random variables and that, therefore, the population includes subpopulations of cells with different distributions for the heat resistance parameters. A linear relationship between the variability of the log of the final bacterial concentration and the logarithmic reduction in the size of the bacterial population was found.

Effects of electron beam irradiation on the variability in survivor number and duration of lag phase of four food-borne organisms.

The effect of electron beam irradiation on microbial inactivation and duration of lag time of individual surviving cells of Listeria innocua, Enterococcus faecalis, Pseudomonas fluorescens and Salmonella Enteritidis has been studied. In addition, the data on variability in microbial inactivation and duration of lag phase for surviving microbes have been fitted by normal and gamma distributions, respectively. The standard deviations of survivor number and lag phase duration of individual cells were higher in irradiated batches than in non-irradiated ones. Furthermore, the more intense the irradiation treatment was, the higher the variability in both survivor number and duration of lag phase of survivors. These findings should be considered in predictive models of microbial inactivation, in risk assessment, and in adjusting preserving and/or storage conditions in the food industry.

A comparison of the effects of E-beam irradiation and heat treatment on the variability of Bacillus cereus inactivation and lag phase duration of surviving cells.

The effects of electron beam irradiation and heat treatments on the variability of inactivation of Bacillus cereus spores (CECT 131/ATCC 10876) and of the lag phase of single surviving cells have been studied. In general, dispersion in the number of survivors increased as the stress became more intense. A polynomial relationship was derived between the coefficient of variation of the survivor number and the inactivation achieved. Heat treatments caused wider distributions than irradiation for the same substrate and for a similar degree of microbial inactivation. Increasing the intensity of the inactivation treatment lengthened the lag phase of survivors and increased its variability. Comparison of lag phases of heated and irradiated spores did not show any clear relationship. Heating did not affect the specific growth rate of surviving cells, whereas irradiation lowered the maximum specific growth rate in proportion to the dose applied. These results suggest that the shelf life of irradiated foods is longer than that of heated foods.

Modeling the Listeria innocua micropopulation lag phase and its variability

Listeria innocua micropopulation lag phase and its variability have been modeled as a function of growth temperature, intensity of heat stress, and the number of surviving cells initiating growth. Micropopulation lag phases were found to correlate negatively with inoculum size and growth temperature and positively with heat shock intensity. Validation of the models using experimental milk samples indicated that the average lag phase duration predicted is shorter and more variable than the observed, meaning that they should be considered safe for risk assessment. Our results suggest that the effect of inoculum size on the population lag phase has both stochastic and physiological components.

Towards lag phase of microbial populations at growth-limiting conditions: The role of the variability in the growth limits of individual cells.

The water activity (aw) growth limits of unheated and heat stressed Listeria monocytogenes individual cells were studied. The aw limits varied from 0.940 to 0.997 and 0.951 to 0.997 for unheated and heat stressed cells, respectively. Due to the above variability a decrease in aw results in the presence of a non-growing fraction in the population leading to an additional pseudo-lag in population growth. In this case the total apparent lag of the population is the sum of the physiological lag of the growing cells (time required to adjust to the new environment) and the pseudo-lag. To investigate the effect of aw on the above lag components, the growth kinetics of L. monocytogenes on tryptone soy agar with aw adjusted to values ranging from 0.997 to 0.940 was monitored. The model of B&R was fitted to the data for the estimation of the apparent lag. In order to estimate the physiological lag of the growing fraction of the inoculum, the model was refitted to the growth data using as initial population level the number of cells that were able to grow (estimated from the number of colonies formed on the agar at the end of storage) and excluding the rest data during the lag. The results showed that for the unheated cells the apparent lag was almost identical to the physiological lag for aw values ranging from 0.997 to 0.970, as the majority of the cells in the initial population was able to grow in these conditions. As the aw decreased from 0.970 to 0.940 however, the number of cells in the population which were able to grow, decreased resulting to an increase in the pseudo-lag. The maximum value of pseudo-lag was 13.1h and it was observed at aw=0.940 where 10% of the total inoculated cells were able to grow. For heat stressed populations a pseudo-lag started to increase at higher aw conditions (0.982) compared to unheated cells. In contrast to the apparent lag, a linear relation between physiological lag and aw was observed for both unheated and heat stressed cells.

Estimation of the growth kinetic parameters of Bacillus cereus spores as affected by pulsed light treatment.

Quantitative microbial risk assessment requires the knowledge of the effect of food preservation technologies on the growth parameters of the survivors of the treatment. This is of special interest in the case of the new non-thermal technologies that are being investigated for minimal processing of foods. This is a study on the effect of pulsed light technology (PL) on the lag phase of Bacillus cereus spores surviving the treatment and the maximum growth rate (μmax) of the survivors after germination. The D value was estimated as 0.35 J/cm(2) and our findings showed that PL affected the kinetic parameters of the microorganism. A log linear relationship was observed between the lag phase and the intensity of the treatment. Increasing the lethality lengthened the mean lag phase and proportionally increased its variability. A polynomial regression was fitted between the μmax of the survivors and the inactivation achieved. The μmax decreased as intensity increased. From these data, and their comparison to published results on the effect of heat and e-beam irradiation on B. cereus spores, it was observed that the shelf-life of PL treated foods would be longer than those treated with heat and similar to irradiated ones. These findings offer information of interest for the implementation of PL for microbial decontamination in the food industry.

Effect of nitrate and nitrite on Listeria and selected spoilage bacteria inoculated in dry-cured ham

The effect of nitrate and the combination of nitrate/nitrite on Listeria innocua (as surrogate of Listeria monocytogenes). And two selected spoilage microorganisms (Proteus vulgaris and Serratia liquefaciens) was studied in dry-cured ham. Hams were manufactured with different concentrations of curing agents: KNO3 (600 and 150mg/kg) alone or in combination with NaNO2 (600 and 150mg/kg). The addition of 500mg/kg of sodium ascorbate was also evaluated in a batch with 600mg/kg of nitrate and nitrite. The target microorganisms were inoculated by injection in semimembranosus, biceps femoris and in the shank, prior to curing. P. vulgaris and S. liquefaciens were controlled by temperature and aw, respectively, and no effect of nitrate/nitrite was observed. The presence of nitrite in the curing mix reduced L. innocua in semimembranosus, which population was 1.5logcfu/g lower at the end of resting (p<0.05), while at the end of the process it was more frequently detected in the no- and low-nitrite added hams. None of the treatments was able to control Listeria in deeper areas of ham. The addition of sodium ascorbate to the curing mix containing the highest amount of nitrate and nitrite did not show any effect on the microorganisms studied.

Characterization of damage on Listeria innocua surviving to pulsed light: Effect on growth, DNA and proteome

The effect of pulsed light treatment on the lag phase and the maximum specific growth rate of Listeria innocua was determined in culture media at 7 °C. Fluences of 0.175, 0.350 and 0.525 J/cm2 were tested. The lag phase of the survivors increased as fluence did, showing significant differences for all the doses; an 8.7-fold increase was observed at 0.525 J/cm2. Pulsed light decreased the maximum specific growth rate by 38% at the same fluence. Both parameters were also determined by time-lapse microscopy at 25 °C in survivors to 0.525 J/cm2, with an increase of 13-fold of the lag phase and a 45% decrease of the maximum specific growth rate. The higher the fluence, the higher the variability of both parameters was. To characterize pulsed light damage on L. innocua, the formation of dimers on DNA was assessed, and a proteomic study was undertaken. In cells treated with 0.525 J/cm2, cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts were detected at 5:1 ratio. Pulsed light induced the expression of three proteins, among them the general stress protein Ctc. Furthermore, treated cells showed an up-regulation of proteins related to metabolism of nucleotides and fatty acids, as well as with translation processes, whereas flagellin and some glucose metabolism proteins were down-regulated. Differences in the proteome of the survivors could contribute to explain the mechanisms of adaptation of L. innocua after pulsed light treatment.