Relative Lag Times Research Articles

Antimicrobial effect of nisin in processed cheese - Quantification of residual nisin by LC-MS/MS and development of new growth and growth boundary model for Listeria monocytogenes

This study tested the hypothesis that growth of Listeria monocytogenes in processed cheese with added nisin can be predicted from residual nisin A concentrations in the final product after processing. A LC-MS/MS method and a bioassay were studied to quantify residual nisin A concentrations and a growth and growth boundary model was developed to predict the antilisterial effect in processed cheese. 278 growth rates were determined in broth for 11 L. monocytogenes isolates and used to determine 13 minimum inhibitory concentration (MIC) values for nisin between pH 5.5 and 6.5. To supplement these data, 67 MIC-values at different pH-values were collected from the scientific literature. A MIC-term was developed to describe the effect of pH on nisin MIC-values. An available growth and growth boundary model (doi: https://doi.org/10.1016/j.fm.2019.103255) was expanded with the new MIC-term for nisin to predict growth in processed cheese. To generate data for model evaluation and further model development, challenge tests with a total of 45 growth curves, were performed using processed cheese. Cheeses were formulated with 11.2 or 12.0 ppm of nisin A and heat treated to obtain residual nisin A concentrations ranging from 0.56 to 5.28 ppm. Below 15 °C, nisin resulted in extended lag times. A global regression approach was used to fit all growth curves determined in challenge tests. This was obtained by combining the secondary growth and growth boundary model including the new term for the inhibiting effect of nisin on μmax with the primary logistic growth model with delay. This model appropriately described the growth inhibiting effect of residual nisin A and showed that relative lag times depended on storage temperatures. With residual nisin A concentrations, other product characteristics and storage temperature as input the new model correctly predicted all observed growth and no-growth responses for L. monocytogenes. This model can support development of nisin A containing recipes for processed cheese that prevent growth of L. monocytogenes. Residual nisin A concentrations in processed cheese were accurately quantified by the developed LC-MS/MS method with recoveries of 83 to 110% and limits of detection and quantification being 0.04 and 0.13 ppm, respectively. The tested bioassay was less precise and nisin A recoveries varied for 53% to 94%.

Growth of Staphylococcus aureus in cooked ready-to-eat ground fish as affected by inoculum size and potassium sorbate as food preservative

This study was conducted to investigate the influence of inoculum size and potassium sorbate on kinetic growth of Staphylococcus aureus in cooked ready-to-eat ground fish (CGF) prevalent retail storage temperatures. Two treatment levels (with or without 0.2% potassium sorbate) and three inoculum sizes (3.0, 4.5, or 5.5 log CFU/g) were evaluated. CGF samples were inoculated and subsequently stored at 10, 15, 20, 25, 30, and 37 °C. Growth curves at each storage temperature were fitted to Baranyi model to estimate lag time (λ, hour) and maximum growth rate (log CFU/g/h). Prediction of λ was dependent on the inoculum size and potassium sorbate treatment. Two secondary λ models (relative lag time (RLT) and hyperbola) were used to describe the variability in the growth behavior of S. aureus on CGF. RLT values were higher for treated samples (3.55, 3.16, and 2.74, respectively for 3 inoculum sizes) than those obtained from untreated samples (3.49, 3.01, and 2.34) and decreased with increasing the inoculum size. Hyperbola model indicated considerable variability but no systematic trend was observed. Relative error within acceptable prediction zone was higher than 80% for overall secondary models developed. This supported that the developed models demonstrated acceptable performance under examined temperatures.

Modelling and predicting growth of psychrotolerant pseudomonads in milk and cottage cheese

Mathematical models were developed and evaluated for growth of psychrotolerant pseudomonads in chilled milk and in cottage cheese with cultured cream dressing. The mathematical models include the effect of temperature, pH, NaCl, lactic acid and sorbic acid. A simplified cardinal parameter growth rate model was developed based on growth in broth. Subsequently, the reference growth rate parameter μref25°C-broth of 1.031/h was calibrated by fitting the model to a total of 35 growth rates from cottage cheese with cultured cream dressing. This resulted in a μref25°C-cottage cheese value of 0.621/h. Predictions from both growth rate models were evaluated by comparison with literature and experimental data. Growth of psychrotolerant pseudomonads in heat-treated milk (n=33) resulted in a bias factor (Bf) of 1.08 and an accuracy factor (Af) of 1.32 (μref25°C-broth), whereas growth in cottage cheese with cultured cream dressing and in non-heated milk (n=26) resulted in Bf of 1.08 and Af of 1.43 (μref25°C-cottage cheese). Lag phase models were developed by using relative lag times and data from both the present study and from literature. The acceptable simulation zone method showed the developed models to successfully predict growth of psychrotolerant pseudomonads in milk and cottage cheese at both constant and dynamic temperature storage conditions. The developed models can be used to predict growth of psychrotolerant pseudomonads and shelf life of chilled cottage cheese and milk at constant and dynamic storage temperatures. The applied methodology and the developed models seem likely to be applicable for shelf life assessment of other types of products where psychrotolerant pseudomonads are important for spoilage.

Stochastic modelling of Listeria monocytogenes single cell growth in cottage cheese with mesophilic lactic acid bacteria from aroma producing cultures

A stochastic model was developed for simultaneous growth of low numbers of Listeria monocytogenes and populations of lactic acid bacteria from the aroma producing cultures applied in cottage cheese. During more than two years, different batches of cottage cheese with aroma culture were analysed for pH, lactic acid concentration and initial concentration of lactic acid bacteria. These data and bootstrap sampling were used to represent product variability in the stochastic model. Lag time data were estimated from observed growth data (lactic acid bacteria) and from literature on L. monocytogenes single cells. These lag time data were expressed as relative lag times and included in growth models. A stochastic model was developed from an existing deterministic growth model including the effect of five environmental factors and inter-bacterial interaction [Østergaard, N.B, Eklöw, A and Dalgaard, P. 2014. Modelling the effect of lactic acid bacteria from starter- and aroma culture on growth of Listeria monocytogenes in cottage cheese. International Journal of Food Microbiology. 188, 15-25]. Growth of L. monocytogenes single cells, using lag time distributions corresponding to three different stress levels, was simulated. The simulated growth was subsequently compared to growth of low concentrations (0.4-1.0CFU/g) of L. monocytogenes in cottage cheese, exposed to similar stresses, and in general a good agreement was observed. In addition, growth simulations were performed using population relative lag time distributions for L. monocytogenes as reported in literature. Comparably good predictions were obtained as for the simulations performed using lag time data for individual cells of L. monocytogenes. Therefore, when lag time data for individual cells are not available, it was suggested that relative lag time distributions for L. monocytogenes can be used as a qualified default assumption when simulating growth of low concentrations of L. monocytogenes.

Analysing and modelling the growth behaviour of Listeria monocytogenes on RTE cooked meat products after a high pressure treatment at 400 MPa

Various predictive models are available for high pressure inactivation of Listeria monocytogenes in food, but currently available models do not consider the growth kinetics of surviving cells during the subsequent storage of products. Therefore, we characterised the growth of L. monocytogenes in sliced cooked meat products after a pressurization treatment. Two inoculum levels (107 or 104CFU/g) and two physiological states before pressurization (freeze-stressed or cold-adapted) were evaluated. Samples of cooked ham and mortadella were inoculated, high pressure processed (400MPa, 5min) and subsequently stored at 4, 8 and 12°C. The Logistic model with delay was used to estimate lag phase (λ) and maximum specific growth rate (μmax) values from the obtained growth curves. The effect of storage temperature on μmax and λ was modelled using the Ratkowsky square root model and the relative lag time (RLT) concept.Compared with cold-adapted cells the freeze-stressed cells were more pressure-resistant and showed a much longer lag phase during growth after the pressure treatment. Interestingly, for high-pressure inactivation and subsequent growth, the time to achieve a concentration of L. monocytogenes 100-fold (2-log) higher than the cell concentration prior to the pressure treatment was similar for the two studied physiological states of the inoculum. Two secondary models were necessary to describe the different growth behaviour of L. monocytogenes on ready-to-eat cooked ham (lean product) and mortadella (fatty product). This supported the need of a product-oriented approach to assess growth after high pressure processing. The performance of the developed predictive models for the growth of L. monocytogenes in high-pressure processed cooked ham and mortadella was evaluated by comparison with available data from the literature and by using the Acceptable Simulation Zone approach. Overall, 91% of the relative errors fell into the Acceptable Simulation Zone.

Global genome response of Escherichia coli O157∶H7 Sakai during dynamic changes in growth kinetics induced by an abrupt temperature downshift.

Escherichia coli O157∶H7 is a mesophilic food-borne pathogen. We investigated the growth kinetics of E. coli O157∶H7 Sakai during an abrupt temperature downshift from 35°C to either 20°C, 17°C, 14°C or 10°C; as well as the molecular mechanisms enabling growth after cold stress upon an abrupt downshift from 35°C to 14°C in an integrated transcriptomic and proteomic analysis. All downshifts caused a lag period of growth before growth resumed at a rate typical of the post-shift temperature. Lag and generation time increased with the magnitude of the shift or with the final temperature, while relative lag time displayed little variation across the test range. Analysis of time-dependent molecular changes revealed, in keeping with a decreased growth rate at lower temperature, repression of genes and proteins involved in DNA replication, protein synthesis and carbohydrate catabolism. Consistent with cold-induced remodelling of the bacterial cell envelope, alterations occurred in the expression of genes and proteins involved in transport and binding. The RpoS regulon exhibited sustained induction confirming its importance in adaptation and growth at 14°C. The RpoE regulon was transiently induced, indicating a potential role for this extracytoplasmic stress response system in the early phase of low temperature adaptation during lag phase. Interestingly, genes previously reported to be amongst the most highly up-regulated under oxidative stress were consistently down-regulated. This comprehensive analysis provides insight into the molecular mechanisms operating during adaptation of E. coli to growth at low temperature and is relevant to its physiological state during chilling in foods, such as carcasses.

MudPIT profiling reveals a link between anaerobic metabolism and the alkaline adaptive response of Listeria monocytogenes EGD-e.

Listeria monocytogenes is a foodborne human pathogen capable of causing life-threatening disease in susceptible populations. Previous proteomic analysis we performed demonstrated that different strains of L. monocytogenes initiate a stringent response when subjected to alkaline growth conditions. Here, using multidimensional protein identification technology (MudPIT), we show that in L. monocytogenes EGD-e this response involves an energy shift to anaerobic pathways in response to the extracellular pH environment. Importantly we show that this supports a reduction in relative lag time following an abrupt transition to low oxygen tension culture conditions. This has important implications for the packaging of fresh and ready-to-eat foods under reduced oxygen conditions in environments where potential exists for alkaline adaptation.

Preparation and evaluation of novel metronidazole sustained release and floating matrix tablets

In the present study, metronidazole was used for preparing floating dosage forms that are designed to retain in the stomach for a long time and have developed as a drug delivery system for better eradication of Helicobacter Pylori in peptic ulcer diseases. For this means, various formulations were designed using multi-factorial design. HPMC, psyllium and carbopol in different concentrations were used as floating agents, and sodium bicarbonate was added as a gas-forming agent. Hardness, friability, drug loading, floating ability and release profiles as well as kinetics of release were assessed. Formulations containing HPMC as filler showed prolonged lag times for buoyancy. Adding psyllium to these formulations had reduced relative lag times. Overall, selected formulations were able to float immediately and showed buoyancy for at least 8 h. Meanwhile, sustained profiles of drug release were also obtained. Kinetically, among the 10 assessed models, the release pattern of metronidazole from the tablets fitted best to Power law, Weibull and Higuchi models in respect overall to mean percentage error values of 3.8, 4.73 and 5.77, respectively, for calcium carbonate-based tablets and, 2.95, 6.39 and 3.9, respectively, for calcium silicate-based tablets. In general, these systems can float in the gastric condition and control the drug release from the tablets.

Modeling and Predicting the Growth of Lactic Acid Bacteria in Lightly Preserved Seafood and Their Inhibiting Effect on Listeria monocytogenes

A cardinal parameter model was developed to predict the effect of diacetate, lactate, CO2, smoke components (phenol), pH, NaCl, temperature, and the interactions between all parameters on the growth of lactic acid bacteria (LAB) in lightly preserved seafood. A product-oriented approach based on careful chemical characterization and growth of bacteria in ready-to-eat seafoods was used to develop this new LAB growth model. Initially, cardinal parameter values for the inhibiting effect of diacetate, lactate, CO2, pH, and NaCl–water activity were determined experimentally for a mixture of LAB isolates or were obtained from the literature. Next, these values and a cardinal parameter model were used to model the effect of temperature (Tmin) and smoke components (Pmax). The cardinal parameter model was fitted to data for growth of LAB (μmax values) in lightly preserved seafood including cold-smoked and marinated products with different concentrations of naturally occurring and added organic acids. Separate product validation studies of the LAB model resulted in average bias and accuracy factor values of 1.2 and 1.5, respectively, for growth of LAB (μmax values) in lightly preserved seafood. Interaction between LAB and Listeria monocytogenes was predicted by combining the developed LAB model and an existing growth and growth boundary model for the pathogen (O. Mejlholm and P. Dalgaard, J. Food Prot. 70:70–84). The performance of the existing L. monocytogenes model was improved by taking into account the effect of microbial interaction with LAB. The observed and predicted maximum population densities of L. monocytogenes in inoculated lightly preserved seafoods were 4.7 and 4.1 log CFU g−1, respectively, whereas for naturally contaminated vacuum-packed cold-smoked salmon the corresponding values were 0.7 and 0.6 log CFU g−1 when a relative lag time of 4.5 was used for the pathogen.

Biogenic volatile organic compounds as potential carbon sources for microbial communities in soil from the rhizosphere ofPopulus tremula

Catabolism of a (14)C-labelled volatile monoterpene compound (geraniol) to (14)CO(2) was investigated in soils taken from the rhizosphere at distances up to 200 cm from the trunks of three small Populus tremula trees growing at different sites in Slovenia. Emissions of limonene of up to 18 microg m(-2) h(-1) were detected from the soil surface at each site. Evolution of (14)C-labelled CO(2) was measured as a product of catabolism of (14)C-labelled geraniol introduced into the soil samples. Indigenous soil microorganisms degraded the geraniol rapidly. There was a significant difference in relative lag times and rates of catabolism along the gradient from the tree trunks, with relatively longer lag times and lower rates occurring in soil samples from the farthest point from the tree.

Viable count estimates of lag time responses for Salmonella typhimurium M48 subjected to abrupt osmotic shifts

Generally, relative lag times (RLT; lag time divided by generation time) become extended as conditions become less favourable for growth. Mellefont et al. (2003, 2004) [Mellefont, L.A., McMeekin, T.A., Ross, T., 2003. The effect of abrupt osmotic shifts on the lag phase duration of foodborne bacteria. Int. J. Food Microbiol. 83(3), 281–293; Mellefont, L.A., McMeekin, T.A., Ross, T., 2004. The effect of abrupt osmotic shifts on the lag phase duration of physiologically distinct populations of Salmonella typhimurium. Int. J. Food Microbiol. 92, 111–120] reported that abrupt osmotic shifts of Salmonella typhimurium M48 from optimal to low a w led to unexpectedly small RLTs at low a w. In this study, RLTs resulting from similar osmotic shifts were estimated by viable count (VC) and compared to turbidimetric estimates to test the hypothesis that the ‘downturn’ in RLT is an artefact of the turbidimetric technique used. No ‘downturn’ in RLT was observed with VC data and RLTs increased with increasing magnitude of osmotic shift. Anomalous turbidimetric estimates of lag time at low a w were confirmed as the likely source of the ‘downturn’ in RLT. The abrupt osmotic shifts resulted in a complex pattern of microbial population behaviour. Immediately after transfer from optimal a w to low a w, inactivation of a portion of the population occurred for all the conditions tested. The degree of inactivation became progressively larger with larger shifts in a w. The initial decline in population was followed by a period during which no change in numbers occurred, followed by growth that appeared, in most cases, to be exponential. At the lowest a ws tested (≤ 0.954), the growth response after the initial decline was at a rate slower than that of exponential phase growth. Due to the use of non-selective media containing pyruvate (to eliminate oxygen radicals), the observed patterns of inactivation, lag and regrowth at most a w conditions are unlikely to result from a temporary loss of culturability, but may represent inactivation of a portion of the population.

Growth of Listeria monocytogenes as influenced by viscosity and water activity

The effects of osmotic stress on Listeria monocytogenes growth parameters was examined in relation to the viscosity of the growth media. In low-viscosity systems, growth of L. monocytogenes in glucose-supplemented media was comparable to growth in sucrose-supplemented media. The relative lag time (RLT: the lag time divided by the generation time) responses were found to increase in the more restrictive water activity conditions. In high-viscosity systems containing polyvinylpyrrolidone (PVP), growth rate was reduced, whereas lag time showed no discernible modification. Osmotic stress in medium- and high-viscosity media supplemented with glucose resulted in approximately exponential increasing of the RLT values. Thus, the biological effects of osmotic stress on L. monocytogenes could be affected by the physical properties of the system, such as viscosity and diffusivity.

The effect of abrupt osmotic shifts on the lag phase duration of physiologically distinct populations of Salmonella typhimurium

Relative lag time (RLT), i.e. lag time divided by generation time, was used to characterise the lag phase response of exponential and stationary phase Salmonella typhimurium subjected to NaCl-mediated hyperosmotic shifts. Abrupt hyperosmotic shifts induced lag phases. The RLT, however, varied with the physiological history of the inoculum and the magnitude of the shift. Turbidimetric data showed that exponential phase cells had larger RLTs (up to ∼8 units) than stationary phase cells (up to 2–4 units). Inocula containing exponential and stationary phase cells mixed in known proportions gave intermediate results. For viable count data, there was little difference in RLT between exponential and stationary phase cells. The RLT response determined turbidimetrically was reproducible for exponential phase cells, but less so for stationary phase cells. It is suggested that there may be a lower limit for resolution of RLT, in the range 0–2 units, and that this may account for the lack of reproducibility in RLTs of stationary phase cells. It is hypothesised that stationary phase cells have enhanced resistance to osmotic stress and are able to exploit new growth environments at low a w more rapidly than exponential phase cells, resulting in shorter lag phases. However, the data indicate that turbidimetry may not accurately describe the lag phase response of exponential phase cells subjected to large osmotic shifts. Viable count data is required to investigate this hypothesis further.

The effect of abrupt shifts in temperature on the lag phase duration of Escherichia coli and Klebsiella oxytoca

The effect of temperature of incubation on lag times of two gram-negative foodborne bacteria was investigated. Bacteria were instantaneously transferred between temperatures within and beyond the normal physiological temperature range (NPTR). Abrupt temperature shifts induced lag phases, but the degree of the response was dependent on the direction and magnitude of the shift. Temperature downshifts induced larger relative lag times (RLT; the ratio of lag time to generation time), than equivalent upshifts. The hypothesis of Robinson et al. [Int. J. Food Microbiol. 44 (1998) 83] that lag time can be understood in terms of the amount of work to be done to adjust to new environmental conditions and the rate at which that work is done was supported. Deviation of the reported proportionality between lag time and generation time was observed when late-exponential phase cells were subjected to abrupt temperature shifts from beyond the normal physiological range.

The effect of abrupt osmotic shifts on the lag phase duration of foodborne bacteria

The effects of osmotic environment and inoculum history on lag times were examined. Abrupt osmotic shifts of cultures were found to induce lag phases in a variety of foodborne bacteria. Relative lag times (RLT; the ratio of lag time to generation time) were used to differentiate the effects of the shift from those of the outgrowth environment. In general, osmotic downshifts induced larger RLTs than equivalent upshifts. An observed reduction in RLT at very low a w, however, was unexpected. For an osmotic downshift, differences were observed in the RLT response of the Gram-negative and -positive strains tested. RLTs were usually extended for Gram-negative organisms as conditions became less favourable for growth. In comparison, RLT remained relatively unaffected for Gram-positive organisms. The observations reported in this study demonstrate that lag time can be understood in terms of the amount of work to be done to adjust to new environmental conditions and the rate at which that work is done, and are consistent with known strategies for osmoregulation employed by the various organisms studied.

Determination of mean growth parameters of bacterial colonies immobilized in gelatin gel using a laser gel-cassette scanner

The potential of a laser gel-cassette scanner (LGS) for rapid, quantitative measurement of the effect of environmental factors such as pH, temperature and humectants on the lag and doubling times of microorganisms is explored. The quantitative relationships between the laser light scattering intensity and colony radius, mean lag time and doubling time are analysed and a measurement protocol is formulated and tested using the specific example of the effect of increasing salt concentration of the lag and doubling times of Salmonella typhimurium LT2. It is concluded that the LGS is a potentially valuable tool for the rapid determination of relative lag and doubling times, but that, because of the need for extensive calibration, it is not capable, at least in its present form, of reliably determining absolute values of lag and doubling times without at least one independent viable count measurement.