Carmen Pin

Predictive models as means to quantify the interactions of spoilage organisms

The purpose of this paper is to quantify the interactions of some groups of spoilage organisms that can be usually found in refrigerated meat stored in air, such as: Enterobacteriaceae, Pseudomonas, Acinetobacter, Psychrobacter, Shewanella, Carnobacterium, Lactobacillus, Leuconostoc, Brochothrix and Kurthia spp. The growth of these organisms was studied in the range of temperature 2-11 degrees C and pH 5.2-6.4, which is characteristic of refrigerated meat. The main growth parameters (maximum specific growth rate and lag time) were modelled by multivariate quadratic polynomials of temperature and pH. The interactions of the organisms were analyzed by comparing their growth models obtained in isolation with those obtained in mixture. The difference between the models was quantified by statistical F-values which were used to measure how much the growth of an organism or group of organisms was affected by others and which of them dominated their joint growth.

Effect of Modified Atmosphere Composition on the Metabolism of Glucose by Brochothrix thermosphacta

ABSTRACT The influence of atmosphere composition on the metabolism of Brochothrix thermosphacta was studied by analyzing the consumption of glucose and the production of ethanol, acetic and lactic acids, acetaldehyde, and diacetyl-acetoin under atmospheres containing different combinations of carbon dioxide and oxygen. When glucose was metabolized under oxygen-free atmospheres, lactic acid was one of the main end products, while under atmospheres rich in oxygen mainly acetoin-diacetyl was produced. The proportions of the total consumed glucose used for the production of acetoin (aerobic metabolism) and lactic acid (anaerobic metabolism) were used to decide whether aerobic or anaerobic metabolism predominated at a given atmosphere composition. The boundary conditions between dominantly anaerobic and aerobic metabolisms were determined by logistic regression. The metabolism of glucose by B. thermosphacta was influenced not only by the oxygen content of the atmosphere but also by the carbon dioxide content. At high CO2 percentages, glucose metabolism remained anaerobic under greater oxygen contents.

Kinetics of Single Cells: Observation and Modeling of a Stochastic Process

ABSTRACT The successive generation times for single cells of Escherichia coli K-12 were measured as described by A. Elfwing, Y. LeMarc, J. Baranyi, and A. Ballagi (Appl. Environ. Microbiol. 70:675-678, 2004), and the histograms they generated were used as empirical distributions to simulate growth of the population as the result of the multiplication of its single cells. This way, a stochastic birth model in which the underlying distributions were measured experimentally was simulated. To validate the model, analogous bacterial growth curves were generated by the use of different inoculum levels. The agreement with the simulation was very good, proving that the growth of the population can be predicted accurately if the distribution of the first few division times for the single cells within that population is known. Two questions were investigated by the simulation. (i) To what extent can we say that the distribution of the detection time, i.e., the time by which a single-cell-generated subpopulation reaches a detectable level, can be identified with that of the lag time of the original single cell? (ii) For low inocula, how does the inoculum size affect the lag time of the population?

Heterogeneity of Times Required for Germination and Outgrowth from Single Spores of Nonproteolytic Clostridium botulinum

ABSTRACT Knowledge of the distribution of growth times from individual spores and quantification of this biovariability are important if predictions of growth in food are to be improved, particularly when, as for Clostridium botulinum, growth is likely to initiate from low numbers of spores. In this study we made a novel attempt to determine the distributions of times associated with the various stages of germination and subsequent growth from spores and the relationships between these stages. The time to germination (tgerm), time to emergence (temerg), and times to reach the lengths of one (tC1) and two (tC2) mature cells were quantified for individual spores of nonproteolytic C. botulinum Eklund 17B using phase-contrast microscopy and image analysis. The times to detection for wells inoculated with individual spores were recorded using a Bioscreen C automated turbidity reader and were compatible with the data obtained microscopically. The distributions of times to events during germination and subsequent growth showed considerable variability, and all stages contributed to the overall variability in the lag time. The times for germination (tgerm), emergence (temerg − tgerm), cell maturation (tC1 − temerg), and doubling (tC2 − tC1) were not found to be correlated. Consequently, it was not possible to predict the total duration of the lag phase from information for just one of the stages, such as germination. As the variability in postgermination stages is relatively large, the first spore to germinate will not necessarily be the first spore to produce actively dividing cells and start neurotoxin production. This information can make a substantial contribution to improved predictive modeling and better quantitative microbiological risk assessment.

Effect of dietary linseed oil and α-tocopherol on pork tenderloin (Psoas major) muscle

The effect of linseed oil and α-tocopheryl acetate on the fatty acid composition and the susceptibility to oxidation of lipid fraction from pork tenderloin (Psoas major) muscle has been studied. Muscles were obtained from animals fed on diets with the same ingredients excepting the oil source [sunflower (C), linseed (L) and linseed and olive (1/1, w/w) (LO)] and α-tocopherol [20 (C, L and LO) or 200 (LOE and LE) mg/kg diet]. The n-6/n-3 ratio in pork tenderloin was markedly modified by dietary linseed oil administration, which was due to the increase in the C18:3n-3 (and total n-3 fatty acids) and the decrease in the C18:2n-6 (and total n-6 fatty acids) contents (P<0.05). The α-tocopherol content of tenderloin from batches LE and LOE was about 2.8 mg/kg of muscle, significantly greater (P<0.05) than about 0.7 mg/kg muscle found in tenderloin from pigs receiving C, L and LO. Dietary supplementation with α-tocopheryl acetate markedly reduced tenderloin lipid oxidation from animals fed diets enriched in n-3 fatty acids (L or LO vs LE or LOE).

Cell surface hydrophobicity and attachment of pathogenic and spoilage bacteria to meat surfaces.

Cell surface hydrophobicity of several pathogenic and spoilage bacteria was determined by hydrocarbon adherence, adhesion to nitrocellulose filter, salt aggregation and adherence to phenyl-sepharose beads. Hydrophobicity of each bacterium was dependent on the method of measurement. Hydrophobicity was compared with the strength of attachment (Sr) of the bacteria to beef muscle surfaces. Bacterial cell surface hydrophobicity, as determined by the bacterial adherence to xylene correlated well with attachment strength (r = 0.800, P < 0.05). Staphylococcus aureus, Clostridium perfringens and Yersinia enterocolitica showed the highest values of attachment strength.

Historical and Contemporary NaCl Concentrations Affect the Duration and Distribution of Lag Times from Individual Spores of Nonproteolytic Clostridium botulinum

ABSTRACT In this study we determined the effect of NaCl concentration during sporulation (0 or 3.0% [wt/vol] added NaCl) and subsequent growth (0 or 2.0% [wt/vol] added NaCl) on the distributions of times associated with various stages of the lag phase of individual spores of nonproteolytic Clostridium botulinum strain Eklund 17B. The effects of NaCl on the probability of germination and the probability of subsequent growth were also determined. Spore populations exhibited considerable heterogeneity at all stages of lag phase for each condition tested. Germination time did not correlate strongly with the times for later stages in the lag phase, such as outgrowth and doubling time. Addition of NaCl to either the sporulation or growth media increased the mean times for, and variability of, all the measured stages of the lag phase (germination, emergence, time to one mature cell, and time to first doubling). There was a synergistic interaction between the inhibitory effects of NaCl in the sporulation medium and the inhibitory effects of NaCl in the subsequent growth medium on the total lag time and each of its stages. Addition of NaCl to either the sporulation medium or the growth medium reduced both the probability of germination and the probability of a germinated spore developing into a mature cell, but the interaction was not synergistic. Spores formed in medium with added NaCl were not better adapted to subsequent growth in suboptimal osmotic conditions than spores formed in medium with no added NaCl were. Knowledge of the distribution of lag times for individual spores and quantification of the biovariability within lag time distributions may provide insight into the underlying mechanisms and can be used to improve predictions of growth in food and to refine risk assessments.

Single-Cell and Population Lag Times as a Function of Cell Age

ABSTRACT After inoculation, the times to the first divisions are longer and more widely distributed for those Escherichia coli single cells that spent more time in the stationary phase prior to inoculation. The second generation times are still longer than the typical generation times in the exponential phase, and this extended the apparent lag time of the cell population. The greater the variability of the single-cell interdivision intervals, the shorter are both the lag time and the doubling time of the population.

Predictive model for the growth of Yersinia enterocolitica under modified atmospheres

A quadratic response surface model is presented to describe the maximum specific growth rate of Yersinia enterocolitica, at refrigeration temperatures, under modified atmospheres. The presence of CO2 affected mainly the lag phase of the organism. The length of the lag phase increased with higher levels of CO2 in the atmosphere, and this effect was more noticeable at low temperatures. The effect of oxygen was similar but less pronounced. The observed growth was slower with higher CO2. Oxygen also decreased the growth rate, but its effect was significant only when its proportion in the atmosphere was greater than about 40%. Model predictions were compared with growth rates obtained in sea food inoculated with Y. enterocolitica and packaged under modified atmospheres. Predictions were also checked to determine whether they were inside the strict interpolation region of the model.

Stress- and Growth Rate-Related Differences between Plate Count and Real-Time PCR Data during Growth of Listeria monocytogenes

ABSTRACT To assess the overestimation of bacterial cell counts in real-time PCR in relation to stress and growth phase, four different strains of L. monocytogenes were exposed to combinations of osmotic stress (0.5 to 8% [vol/vol] NaCl) and acid stress (pH 5 to 7) in a culture model at a growth temperature of 10°C or were grown under optimal conditions. Growth curves obtained from real-time PCR, optical density, and viable count data were compared. As expected, optical density data revealed entirely different growth curves. Good to moderate growth conditions yielded good correlation of real-time PCR data and plate count data (r2 = 0.96 and 0.99) with similar cell counts. When growth conditions became worse, the numbers of CFU decreased during the stationary phase, whereas real-time-PCR-derived bacterial cell equivalents differed in this regard; the correlation worsened (r2 = 0.84). However, fitted growth curves revealed that maximum growth rates calculated from real-time PCR data were not significantly different from those derived from plate count data. The overestimation of bacterial cell counts by real-time PCR observed in the stationary phase under higher-stress conditions might be explained by the accumulation of viable but nonculturable bacteria or dead bacteria and extracellular DNA. Considering these results, real-time PCR data collected from naturally contaminated samples should be viewed with caution.