Germination at different water activities of similarly aged Metarhizium conidia harvested from ageing cultures

The ability of two Metarhizium species, M. acridum (IMI 330189) and M. anisopliae (IMI 385045), to germinate at five water activities ranging from 0.998 to 0.944, was examined for conidia harvested from cultures of different ages. The culture ages ranged from 4 to 22 days old. All of the conidia on each plate were harvested 4 days after inoculation, then again after 11, 18 and 22 days. Therefore, the conidia were never greater than 7 days old. The data suggested that conidia of M. anisopliae were generally fitter, as defined by higher viability at all water activities, when harvested from the 4 day-old culture compared to those from the 22 day-old culture. At the lowest water activity (0.944) germination of M. anisopliae conidia was 71.5% from the 4 day-old culture compared to 0% from the 22 day-old culture. In contrast, conidia of M. acridum were generally fitter when they were obtained from 11, 18 and 22 day-old cultures than the conidia obtained from the 4 day-old culture. However, at the lowest water activity, germination for all treatments of M. acridum was less than 8%. The culture growth phase and media age may affect conidia fitness. The capacity of conidia to withstand water stress and possible reasons for interspecific variation, including susceptibility to imbibitional damage, are discussed.     

Modelling the effect of temperature and water activity on the growth of Aspergillus parasiticus on irradiated Argentinian flint maize

A full factorially designed experiment including storage temperature (10, 20, 30 and 37 °C) and water activity (0.88, 0.92 and 0.96) was undertaken to study the growth of Aspergillus parasiticus in maize samples. Kinetic parameters such as specific growth rate (μ), lag phase duration and maximum logarithmic increase were determined by fitting the Modified Gompertz equation to the viable mould count data (N in CFU/g) as a function of time collected in twelve experiments. The average coefficient of determination (R²) was 0.987, being the mean standard deviation of the estimate of 0.216 in units of log₁₀N. In the practical range of 10-30 °C, the relationship of the three kinetic parameters with temperature was described by second order polynomial expressions, whose parameters, in turn, depended on water activity. The combined or full model i.e., the Modified Gompertz model with its parameters expressed as a function of temperature and water activity, was able to predict log₁₀N with an average percentage error of 4.3, so agreement with the experimental data was highly satisfactory.In a simulation exercise, the full model was able to predict the viable mould count, given an initial value and grain temperature and water activity histories, with promising results for maize storage.     

Modelling the effect of temperature and water activity on the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea

The purpose of the present study was to apply a modelling approach to define the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea on a synthetic medium as a function of temperature and water activity. Both fungal species were grown on malt extract agar at different temperatures (10, 15, 20, 25, 30, 35, 40 and 45 °C) and a_w levels (0.88, 0.90, 0.92, 0.94, 0.96 and 0.99) for a period of 30 days. Growth responses were evaluated over time in terms of colony diameter changes. Growth data were fitted to the primary model of Baranyi and the resulting growth rates were further modeled as a function of temperature and water activity using the cardinal model with inflection (CMI) ( Rosso et al., 1993). A logistic regression quadratic polynomial model was also employed to predict the probability of growth over storage time. Estimated parameters for minimum, maximum and optimum temperatures for growth were 9.1 °C, 46.4 °C and 32.1 °C for B. fulva and 10.5 °C, 43.2 °C and 32.1 °C for B. nivea. The respective values for a_w were 0.893, 0.993 and 0.985 for B. fulva and 0.892, 0.992 and 0.984 for B. nivea. No growth was observed at 0.88 a_w regardless of temperature for both species, whereas B. nivea ascospores could not grow at 10 and 45 °C irrespective of a_w. Regarding growth boundaries, the degree of agreement between predictions and observations was >98% concordant for both species. The erroneously predicted growth cases were 1.4–4.2% false positive and 2.1–3.5% false negative for B. nivea and B. fulva, respectively. The developed logistic model was validated with two literature data sets as well as with data from independent experiments carried out on fruit juices. Validation results showed that agreement with literature data for growth was 25 out of 36 (69.4%) cases, whereas validation on fruit juice data failed in only 6 cases (5 false positives and 1 false negative) out of 128 cases.     

Effect of water activity, temperature and incubation time on growth and ochratoxin A production by Aspergillus niger and Aspergillus carbonarius on maize kernels

The aim of this study was to determine the effects of water activity (a_w) (0.92-0.98), temperature (5-45 °C) and incubation time (5-60 days) on growth and ochratoxin A (OTA) production by Aspergillus niger and Aspergillus carbonarius on maize kernels using a simple method. Colony diameters of both strains at 0.92 a_w were significantly lower than those at 0.96 and 0.98 a_w levels. The optimum growth temperature range for A. niger was 25-40 °C and for A. carbonarius 20-35 °C. A. niger produced OTA from 15 to 40 °C, and the highest OTA level was recorded at 15 °C. The concentration of OTA produced at 0.92 a_w was significantly lower than those at 0.96 and 0.98 a_w. A. carbonarius produced OTA from 15 to 35 °C and the maximum concentration was achieved at 15 °C, although not differing statistically from the concentration detected at 20 °C. At 0.98 a_w the OTA concentration was significantly higher than at 0.96 and 0.92 a_w. Our results show that maize supports both growth and OTA production by A. niger and A. carbonarius. The studied strains were able to produce OTA in maize kernels from the fifth day of incubation over a wide range of temperatures and water availabilities. Although the limit of quantification of our method was higher than that required for the analysis of OTA in food commodities, it has proved to be a useful and rapid way to detect OTA production by fungi inoculated onto natural substrates, in a similar way as for pure culture. Both species could be a source of OTA in this cereal in temperate and tropical zones of the world.     

Modelling the effect of temperature and water activity in the growth boundaries of Aspergillus ochraceus and Aspergillus parasiticus

The aim of this work was to model the growth of Aspergillus parasiticus and Aspergillus ochraceus, both mycotoxin producers, near to the growth/no growth boundaries and validate those models in sterile maize grain, peanuts and coffee beans. Malt extract agar was adjusted to six different water activities: 0.93, 0.91, 0.89, 0.87, 0.85 and 0.80. Plates were incubated at 10, 15, 20, 25, 30, 37 and 42 °C. For each of the 42 conditions, 10 Petri dishes were inoculated. Both kinetic and probability models were applied to colony growth data. The results of the present study indicate that the developed probability modelling approach could be satisfactorily employed to quantify the combined effect of temperature and water activity on the growth responses of A. ochraceus and A. parasiticus. However, validation of kinetic results led to poor goodness of prediction. In this study, the validation samples were placed near to the expected boundaries of the models in order to test them under the worst situation. Probability of growth prediction under extreme growth conditions was somewhat compromised, but it can be considered acceptable.     

Modeling the growth rate and lag time of different strains of Salmonella enterica and Listeria monocytogenes in ready-to-eat lettuce

The growth parameters (growth rate, μ and lag time, λ) of three different strains each of Salmonella enterica and Listeria monocytogenes in minimally processed lettuce (MPL) and their changes as a function of temperature were modeled. MPL were packed under modified atmosphere (5% O₂, 15% CO₂ and 80% N₂), stored at 7–30 °C and samples collected at different time intervals were enumerated for S. enterica and L. monocytogenes. Growth curves and equations describing the relationship between μ and λ as a function of temperature were constructed using the DMFit Excel add-in and through linear regression, respectively. The predicted growth parameters for the pathogens observed in this study were compared to ComBase, Pathogen modeling program (PMP) and data from the literature. High R² values (0.97 and 0.93) were observed for average growth curves of different strains of pathogens grown on MPL. Secondary models of μ and λ for both pathogens followed a linear trend with high R² values (>0.90). Root mean square error (RMSE) showed that the models obtained are accurate and suitable for modeling the growth of S. enterica and L. monocytogenes in MP lettuce. The current study provides growth models for these foodborne pathogens that can be used in microbial risk assessment.     

Modelling the effect of environmental factors on the growth of Aspergillus parasiticus and mycotoxin production in paddy during storage

The simulated experiment of A. parasiticus isolated from the paddy was carried out during the paddy storage for 20 days. The growth and mycotoxin data were collected for constructing kinetic and probability models of moulds. The Baranyi and Gompertz model was employed as the primary model and estimated the lag phase and maximum specific growth rate. Secondary models, such as polynomial, Davey and Gibson model were used and completely evaluated under different conditions. The polynomial equation was highly rated compared with Gibson and Davey model and gave realistic temperatures and a_w for mould growth. Logistic model showed promising results on the prediction of growth boundary and AFB₁ production. Employed models showed promising predicted results, indicating that it is an effective tool for describing and predicting the growth of moulds under different temperatures and a_w. The results can be applied to develop the optimal strategy to prevent fungal spoilage and aflatoxin production during paddy storage.     

Lag time variability in individual spores of Clostridium botulinum

Quantifying lag times from individual spores and the associated variability is an important part of understanding the hazard associated with spore-forming pathogens such as Clostridium botulinum. Knowledge of the underlying distribution would allow greater refinement of risk assessments. To date most studies have either examined lag time indirectly by measuring time to growth or have only examined the first stage of lag, germination. Recent studies have attempted to quantify the variability of spores during the different stages of lag phase and to examine the relationships between these stages. The effect of incubation temperature (22 °C, 15 °C, 10 °C or 8 °C), heat treatment (unheated or 80 °C for 20 s) and sodium chloride concentration in both the sporulation medium (0 or 3% w/v) or growth medium (0 or 2% w/v) on growth from individual spores has been examined. These studies found spores within a single population are very heterogeneous with large variability in all stages of lag. The duration and variability of times for germination, outgrowth and first doubling depended on both the historic treatment of the spores and the prevailing growth conditions, and the stage of lag most affected was treatment dependant.     

Modeling the influence of water activity and ascospore age on the growth of Neosartorya fischeri in pineapple juice

The ascospores of resistant fungi, Neosartorya fischeri, can survive commercial pasteurization, diminishing the shelf life of these products. The time that the ascospores remain in the environment and the effect that they can cause on mold growth are still unknown. This study is aimed to evaluate the influence of water activity (a_w) from 0.90 to 0.99 and the ascospore age (I) from 30 to 90 days of vitro incubation on the growth of N. fischeri in pineapple juice by mathematical modeling. The growth parameters on pineapple juice: adaptation phase (λ), maximum specific growth rate (μ_max) and maximum diameter reached by the colony (A) were obtained by fitting Modified Gompertz and Logistic models to the experimental data. Both models were able to describe microbial growth in pineapple juice, but the Modified Gompertz model presented a slightly superior performance based on statistical indices (correlation coefficients (R²), mean square error (MSE), Bias Factor and Accuracy Factor). The minimum values of λ and A, calculated by the Modified Gompertz model, were 64.7 h and 6.3 mm, while the maximum values were 178.2 h and 20.8 mm, respectively. The result showed that ascospore age did not influence the growth but a_w was statistically significant to the growth parameters λ and A.     

Effect of antioxidants and competing mycoflora on Fusarium verticillioides and F. proliferatum populations and fumonisin production on maize grain

This study was carried out to determine the temporal effect of the antioxidants butylated hydroxyanisol (BHA) and propyl paraben (PP) at doses of 500 and 1000 μg/g on the growth of Fusarium verticillioides and F. proliferatum inoculated on natural maize grain in the presence of the competing mycoflora and fumonisin production at 0.98 and 0.95 water activity (a_w) over a 28-day storage period. The reduction in the log colony forming units (CFU) of Penicillium, Aspergillus and Fusarium populations was 10-100 fold depending on dose of BHA or PP, a_w and time. However, the populations of all three groups were higher at 0.98 a_w than 0.95 a_w. BHA at 500 μg/g and 0.95 a_w reduced the fumonisin content by 82% after 7-14 days incubation, but at the end of the experimental period the reduction was only 32%. A higher reduction in the level of fumonisin produced (77%) was achieved with BHA at 1000 μg/g after 28 days. PP at 500 and 1000 μg/g decreased fumonisin production throughout the incubation period in the drier treatment, but at 0.98 a_w control of toxin production was only achieved after 7-14 days. The reduction in the fumonisin levels could be due to the combined effect of antioxidants, and the competing mycoflora, mainly Aspergillus and Penicillium species.     

Physiological traits of Penicillium glabrum strain LCP 08.5568, a filamentous fungus isolated from bottled aromatised mineral water

Penicillium glabrum is a ubiquitous fungus distributed world wide. This fungus is a frequent contaminant in the food manufacturing industry. Environmental factors such as temperature, water activity and pH have a great influence on fungal development. In this study, a strain of P. glabrum referenced to as LCP 08.5568, has been isolated from a bottle of aromatised mineral water. The effects of temperature, a_w and pH on radial growth rate were assessed on Czapeck Yeast Agar (CYA) medium. Models derived from the cardinal model with inflection [Rosso et al., 1993 An unexpected correlation between cardinal temperatures of microbial growth highlighted by a new model. J. Theor. Bio. 162, 447–463.] were used to fit the experimental data and determine for each factor, the cardinal parameters (minimum, optimum and maximum). Precise characterisation of the growth conditions for such a fungal contaminant, has an evident interest to understand and to prevent spoilage of food products.     

Optimization of pressure-induced germination of Bacillus sporothermodurans spores in water and milk

Bacillus sporothermodurans produces highly resistant endospores that can survive ultra-high-temperature treatment in milk. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk sterility. In this work, the rate of spore germination of B. sporothermodurans LTIS27 was measured in distilled water after high-pressure treatments with varying pressure (50–600 MPa), treatment temperature (20–50 °C), pressure-holding time (5–30 min) and post-pressurization incubation time (30–120 min) at 37 °C or 4 °C. The results showed that pressure-induced germination was maximal (62%) after a treatment at 200 MPa and 20 °C and increased with pressure-holding time and post-pressurization incubation time. Treatment temperature had no significant effect on germination. A central composite experimental design with three factors (pressure, pressure-holding time, and post-pressurization incubation time) using response surface methodology was used to optimize the germination rate in distilled water and in skim milk. No factor interaction was observed. Germination was induced at lower pressure and was faster in milk than in distilled water, but complete germination was not reached. The optimum germination obtained with experimental data was 5.0 log cfu/mL in distilled water and 5.2 log cfu/mL in milk from 5.7 log cfu/mL of spores initially present in the suspension. This study shows the potential of using high hydrostatic pressure to induce the germination of B. sporothermodurans spores in milk before a heat treatment.     

Modelling mould growth under suboptimal environmental conditions and inoculum size

Predictive models can be a tool to develop strategies to prevent mould development and consequently mycotoxin production. The aims of this work were to assess the impact of a) high/low levels of inoculum and b) optimal/suboptimal environmental conditions on fungal responses based on both kinetic and probabilistic models. Different levels of spore suspensions of Aspergillus carbonarius and Penicillium expansum were prepared and inoculated centrally with a needlepoint load on malt extract agar (MEA) with 50 replicates. While optimum conditions led to a colony diameter increase which followed Baranyi’s function, suboptimal conditions led to different grow functions. In general, growth rate (μ) and lag phase (λ) were normally distributed. Specifically, the growth rate (μ) showed similar distributions under optimal growth conditions, regardless of the inoculum level, while suboptimal a_w and temperature conditions led to higher kurtosis distributions, mainly when the inoculum levels were low. Regarding λ, more skewed distributions were observed, mainly when the inoculum levels were low. Probability models were not much affected by the inoculum size. Lower probabilities of growth were in general predicted under marginal conditions at a given time for both strains. The slopes of the probability curves were smaller under suboptimal growth conditions due to wider distributions. Results showed that a low inoculum level and suboptimal conditions lead to high variability of the estimated growth parameters and growth probability.     

Effect of the medium characteristics and the heating and cooling rates on the nonisothermal heat resistance of Bacillus sporothermodurans IC4 spores

In recent years, highly thermo-resistant mesophilic spore-forming bacteria belonging to the species Bacillus sporothermodurans have caused non-sterility problems in industrial sterilization processes. The aim of this research was to evaluate the effect of the heating medium characteristics (pH and buffer/food) on the thermal inactivation of B. sporothermodurans spores when exposed to isothermal and non-isothermal heating and cooling treatments and the suitability of non-linear Weibull and Geeraaerd models to predict the survivors of these thermal treatments. Thermal treatments were carried out in pH 3, 5 and 7 McIlvaine buffer and in a courgette soup. Isothermal survival curves showed shoulders that were accurately characterized by means of both models. A clear effect of the pH of the heating medium was observed, decreasing the D₁₂₀ value from pH 7 to pH 3 buffer down to one third. Differences in heat resistance were similar, regardless of the model used and were kept at all temperatures tested. The heat resistance in courgette soup was similar to that shown in pH 7 buffer. When the heat resistance values obtained under isothermal conditions were used to predict the survival in the non-isothermical experiments, the predictions estimated the experimental data quite accurately, both with Weibull and Geeraerd models.     

Factors affecting growth and pigmentation of Penicillium caseifulvum

Color formation, metabolite production and growth of Penicillium caseifulvum were studied in order to elucidate factors contributing to yellow discoloration of Blue Cheese caused by the mold. A screening experiment was set up to study the effect of pH, concentration of salt (NaCl), P, K, N, S, Mg and the trace metals Fe, Cu, Zn, Mn on yellow color formation, metabolite production and mold growth. Multivariate statistical analysis showed that the most important factor affecting yellow color formation was pH. The most pronounced formation of yellow color, supported by highest amount of colored metabolites, appeared at low pH (pH 4). Mold growth was not correlated to the yellow color formation. Salt concentration was the most important factor affecting mold growth and length of lag phase. Production of secondary metabolites was strongly influenced by both pH and salt concentration. The screening results were used to divide the metabolites into the following three groups: 1) correlated to growth, 2) correlated to color formation, and 3) formed at high pH. Subsequently, a full factorial experiment with factors P, Mg and Cu, showed that low P concentrations (2,000 mg/kg) induced yellow color formation. Among the factors contributing to yellow color formation, pH and salt concentration are easy to control for the cheesemaker, while the third factor, P-concentration, is not. Naturally occurring variations in the P-concentration in milk delivered to Blue Cheese plants, could be responsible for the yellow discoloration phenomenon observed in the dairy industry.     

Influence of the sporulation temperature on the impact of the nutrients inosine and l-alanine on Bacillus cereus spore germination

The effect of sporulation temperature on Bacillus cereus spore germination triggered by the nutrient germinants inosine and l-alanine was investigated. The germination (expressed as % A(600nm) fall) of heat-activated spores of B. cereus strain ATCC14579 produced at 20 and 37 degrees C, and of psychrotrophic strains LM9 and D15 produced at 15 and 37 degrees C was followed in a germination buffer containing inosine at concentrations between 0.01 and 10mmoll(-1), or l-alanine between 1 and 100mmoll(-1). Spores wet-heat resistance at 90 degrees C was also determined. Spores of the three strains produced at the lowest temperatures generally showed a higher germination capacity in response to both inosine and in l-alanine than those produced at 37 degrees C, and were also more susceptible to heat. Low sporulation temperature is confirmed as a detrimental factor to B. cereus spore wet-heat resistance, and conversely gave spores with a sensitivity to the nutrient germinants inosine and l-alanine higher than that of spores formed close to the maximal sporulation temperature.     

The effect of X-ray irradiation on Salmonella inactivation and sensory quality of almonds and walnuts as a function of water activity

The overall goal of this study was to develop a set of process design principles for low-energy X-ray irradiation of tree nuts. Almonds and walnuts were inoculated with Salmonella Enteritidis PT30 and Salmonella Tennessee, and conditioned to four different water activities (0.23, 0.45, 0.64, and 0.84 a_w). Thereafter, the inoculated/conditioned samples were irradiated to achieve up to a 5-log reduction in Salmonella using a pilot scale low-energy X-ray food irradiator. Greater efficacy (D₁₀-value: the dose required to eliminate 90% of the microbial population) for inactivating SE PT30 and S. Tennessee was seen on the surface of almonds (0.226-0.431 kGy) than on walnuts (0.474-0.930 kGy) at all water activities. Also, the efficacy did not change monotonically with water activity. Overall, no significant difference (P > 0.05) in sensory characteristics was seen between non-irradiated almonds and those irradiated to achieve a 5 log reduction in Salmonella. However, irradiating walnuts to the dose corresponding to a 5 log reduction caused a perceivable change in flavor. Post-irradiation storage tests revealed that surviving bacterial counts did not change over 120 days, regardless of nut type, Salmonella serovar, and a_w. Therefore, low-energy X-ray irradiation technology appears to be a promising non-thermal pasteurization strategy for certain types of nuts.     

Effect of water activity and temperature on growth of Alternaria alternata on a synthetic tomato medium

Alternaria alternata is a toxigenic fungus, predominantly responsible for Blackmould of ripe tomato fruits, a disease frequently causing substantial losses of tomatoes, especially those used for canning. The objective of this study was to determine the effect of water activity (a_w, 0.904, 0.922, 0.954, 0.982) and temperature (6, 15, 21 and 35 °C) on germination and radial growth rate on a synthetic tomato medium of a cocktail inoculum of five strains of A. alternata isolated from tomato fruits affected by Blackmould. The shortest germination time (1.5 days) was observed at 0.982 a_w, both at 21 °C and 35 °C. The germination time increased with a reduction on a_w. The fastest growth rate was registered at 0.982 a_w and 21 °C (8.31 mm/day). Growth rates were higher when a_w increased. No growth or germination was observed at the lowest a_w level evaluated (0.904) after 100 days of incubation at 6 °C and 15 °C. A temperature of 6 °C caused a significant reduction in growth rates, even at the optimum a_w level. The knowledge on the ecophysiology of the fungus in this substrate is necessary to elaborate future strategies to prevent its development and evaluate the consumer health risk.     

Effects of copper on germination,growth and sporulation of Clostridium tyrobutyricum

The effects of copper (Cu²⁺) on spore germination, vegetative growth and sporulation of Clostridium tyrobutyricum, which is capable to causing texture and flavour defects in Emmental cheese, were studied. Spore suspensions of three different strains were used as starting material for two experimental set-ups. The first studied the effects of supplemented (0–30 ppm) copper in RCM medium during spore germination and vegetative growth of C. tyrobutyricum measured by plating. The second set-up studied the effects of copper (0–30 ppm) in RCM medium during growth and sporulation of C. tyrobutyricum as measured by optical density at 550 nm and by platings after heat treatment of the samples respectively. Inhibition of germination, vegetative growth and sporulation processes by copper was strain-dependent. Both sporulation and germination were more sensitive than vegetative growth of C. tyrobutyricum to the inhibitory effects of copper. Copper, at the concentrations investigated in this study, inhibits spore germination of C. tyrobutyricum strains. Consequently copper may reduce the risk of late blowing spoilage from in the germination of C. tyrobutyricum spores during the ripening period of Emmental cheese.     

Combined effect of lysozyme and nisin at different incubation temperature and mild heat treatment on the probability of time to growth of Bacillus cereus

Stochastic models can be useful to predict the risk of foodborne illness. The presence of Bacillus cereus in liquid egg can pose a serious hazard to the food industry, since a mild heat treatment cannot guarantee its complete inactivation. However, most of the information available in the scientific literature is deterministic, including growth of B. cereus. In this paper, a stochastic approach to evaluate growth of B. cereus cells influenced by different stresses (presence of nisin and lysozyme separately or in combination) was performed, using an individual-based approach of growth through OD measurements. Lag phase duration was derived from the growth curves obtained. From results obtained, histograms of the lag phase were generated and distributions were fitted. Normal and Weibull distributions were ranked as the bestfit distributions in experiments performed at 25 °C. At 16 °C, lag values (obtained in presence of combinations of both antimicrobials) were also fitted by a Gamma distribution. Predictions were compared with growth curves obtained in liquid egg exposed to mild heat, nisin and/or lysozyme to assess their validity.