Modeling Growth Rate and Assessing Aflatoxins Production by Aspergillus flavus as a Function of Water Activity and Temperature on Polished and Brown Rice

Abstract: The aim of this study was to model the radial growth rate and to assess aflatoxin production by Aspergillus flavus as a function of water activity (a_w 0.82 to 0.92) and temperature (12 to 42 °C) on polished and brown rice. The growth of the fungi, expressed as colony diameter (mm) was measured daily, and the aflatoxins were analyzed using HPLC with a fluorescence detector. The growth rates were estimated using the primary model of Baranyi, which describes the change in colony radius as a function of time. Total of 2 secondary models were used to describe the combined effects of a_w and temperature on the growth rates. The models were validated using independent experimental data. Linear Arrhenius–Davey model proved to be the best predictor of A. flavus growth rates on polished and brown rice followed by polynomial model. The estimated optimal growth temperature was around 30 °C. A. flavus growth and aflatoxins were not detected at 0.82 a_w on polished rice while growth and aflatoxins were detected at this a_w between 25 and 35 °C on brown rice. The highest amounts of toxins were formed at the highest a_w values (0.90 to 0.92) at a temperature of 20 °C after 21 d of incubation on both types of rice. Nevertheless, the consistencies of toxin production within a wider range of a_w values occurred between 25 to 30 °C. Brown rice seems to support A. flavus growth and aflatoxin production more than the polished rice. Practical Application: The developed models can be used to estimate to what extent the change in grain ecosystem conditions affect the storage stability and safety of grains without the need for running long‐standing storage study. By monitoring the intergranular relative humidity and temperature at different locations in the storage facility and inputting these data into the models, it is directly possible to assess either the conditions are conductive for the growth of A. flavus or aflatoxin production.     

Temperature,water activity and gas composition effects on the growth and aflatoxin production by Aspergillus flavus on paddy

The main aim of this study was to evaluate the combined effects of temperature with water activity (a_w) and CO₂ with a_w on the growth and aflatoxin production by Aspergillus flavus Link on paddy. The effects of temperature (20–30 °C) and a_w (0.92–0.98) on the relationship between colony diameter and aflatoxin production, and the influence of a_w (0.92–0.98) and CO₂ (20–80%) on the growth and toxin production were studied using full factorial design. Colony diameters were regularly measured and aflatoxins were periodically analyzed using HPLC with fluorescence detector. The growth and aflatoxin formation increased with a_w at the temperatures studied, and toxin production was positively correlated with the incubation time and colony diameter. Except at 0.92 a_w, as much as 80% CO₂ failed to inhibit the growth of fungi completely. However, at all a_w levels studied the growth parameters as estimated by Baranyi function and aflatoxin were affected by the increment in CO₂ where growth rates and aflatoxin were negatively correlated with CO₂ while the lag phase durations were positively correlated with CO₂. Under 0.98 a_w, the atmosphere enriched with 20% and 80% CO₂ lead to at least 59% and 88% reduction in growth and 47% and 97% in the toxin production, respectively. At 0.95 a_w, the lag phases of both isolates in average increased by a factor of 1.7–12 when the CO₂ levels in the headspace were between 20 and 80% compared to the control. The growth rate and lag phase durations under the modified atmospheres were successfully described using a polynomial equation (R² > 0.97). The results of the study could form a basis of indicative guidelines on the possible control of A. flavus and aflatoxin in paddy during temporary storage prior to drying.     

Influence of water activity and temperature on growth and mycotoxin production by Alternaria alternata on irradiated soya beans

The aim of this study was to determine the effects of water activity (a_w) (0.99-0.90), temperature (15, 25 and 30 °C) and their interactions on growth and alternariol (AOH) and alternariol monomethyl ether (AME) production by Alternaria alternata on irradiated soya beans. Maximum growth rates were obtained at 0.980 a_w and 25 °C. Minimum a_w level for growth was dependent on temperature. Both strains were able to grow at the lowest a_w assayed (0.90). Maximum amount of AOH was produced at 0.98 a_w but at different temperatures, 15 and 25 °C, for the strains RC 21 and RC 39 respectively. Maximum AME production was obtained at 0.98 a_w and 30 °C for both strains. The concentration range of both toxins varied considerably depending on a_w and temperature interactions. The two metabolites were produced over the temperature range 15 to 30 °C and a_w range 0.99 to 0.96. The limiting a_w for detectable mycotoxin production is slightly greater than that for growth. Two-dimensional profiles of a_w × temperature were developed from these data to identify areas where conditions indicate a significant risk from AOH and AME accumulation on soya bean. Knowledge of AOH and AME production under marginal or sub-optimal temperature and a_w conditions for growth can be important since improper storage conditions accompanied by elevated temperature and moisture content in the grain can favour further mycotoxin production and lead to reduction in grain quality. This could present a hazard if the grain is used for human consumption or animal feedstuff.     

Mathematical models to predict kinetic behavior and aflatoxin production of Aspergillus flavus under various temperature and water activity conditions

Mathematical models were developed to predict fungal growth and aflatoxin production of Aspergillus flavus. Fungal growth and aflatoxin concentrations were measured. The Baranyi model was fitted to fungal growth and toxin production data to calculate kinetic parameters. Quadratic polynomial and Gaussian models were then fitted to μ_max and LPD (lag phase duration) values. The ranges of temperature and a _w values showing a μ_max value increase were 15–35°C and 0.891–0.984, respectively. LPD was only observed when the temperature was 20–35°C with a _w=0.891?0.972. The μ_{max growth} value increased up to 35°C with \(b_w = 0.2\left( {b_w = \sqrt {1 - a_w } } \right)\) , then values declined. LPD_growth values increased as the b _w value increased. The μ_max value for aflatoxins increased up to 25°C, but decreased after 30°C, indicating that the developed models are useful for describing the kinetic behavior of Aspergillus flavus growth and aflatoxin production.     

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.     

On the selection of relevant environmental factors to predict microbial dynamics in solidified media

Several studies have shown that food structure causes slower growth rates and narrower growth boundaries of bacteria compared to laboratory media. In predictive microbiology, both a_w or corresponding solute concentration (mainly NaCl) have been used as a growth influencing factor for kinetic models or growth/no growth interface models. The majority of these models have been based on data generated in liquid broth media with NaCl as the predominant a_w influencing solute. However, in complex food systems, other a_w influencing components might be present, next to NaCl. In this study, the growth rate of Salmonella typhimurium was studied in the growth region and the growth/no growth response was tested in Tryptic Soy Broth at 20 °C at varying gelatin concentration (0, 10, 50 g L⁻¹ gelatin), pH (3.25–5.5) and water activity (a_w) (0.929–0.996). From the viewpoint of water activity, the results suggest that NaCl is the main a_w affecting compound. However, gelatin seemed to have an effect on medium a_w too. Moreover, there is also an interaction effect between NaCl and gelatin. From the microbial viewpoint, the results confirmed that the a_w decreasing effect of gelatin is less harmful to cells than the effect of Na⁺ ions. The unexpected shift of the growth/no growth interface to more severe conditions when going from a liquid medium to a medium with 10 g L⁻¹ gelatin is more pronounced when formulating the models in terms of a_w than in terms of NaCl concentrations. At 50 g L⁻¹ gelatin, the model factored with NaCl concentration shifts to milder conditions (concordant to literature results) while the model with a_w indicates a further shift to more severe conditions, which is due to the water activity lowering effect of gelatin and the interaction between gelatin and NaCl. The results suggest that solute concentration should be used instead of a_w, both for kinetic models in the growth region and for growth/no growth interface models, if the transferability of models to solid foods is to be increased.     

Use of response surface methodology in shelf life extension studies of a bakery product

Effects of water activity, pH, storage temperature, concentration of potassium sorbate, CO₂ concentration in the package headspace and inoculum level on growth of Aspergillus niger, the major spoilage isolate of English style crumpets, was studied on an agar model system using an experimental design termed Response Surface Methodology. The initial screening design indicated that a_w, pH, levels of potassium sorbate, CO₂ and storage temperature were significant variables. When a central composite rotatable design was subsequently applied to four variables with pH held constant at pH 6.5 only a_w, storage temperature and CO₂ level were found to have a significant effect on mold growth. Using multiple regression analysis, a second order polynomial model was derived and used to predict the number of days to visible mold growth under various combinations of the remaining three variables. Generation of contour plots enabled selection of levels for each variable to give a product with a desired mold free shelf life. Typically, crumpets with an a_w of 0.96, packaged in 62% CO₂ had a mold free shelf life of 20 d when stored at 20°C. The observed shelf life results agreed well with predicted values on Potato Dextrose Agar and demonstrated RSM as a powerful and elegant research tool when several variables are to be evaluated simultaneously.     

The growth and aflatoxin production of Aspergillus flavus strains on a cheese model system are influenced by physicochemical factors

Traditional cheeses may be contaminated by aflatoxin-producing Aspergillus flavus during the ripening process, which has not been sufficiently taken into account. The objectives of this study were to evaluate the influence of water activity (a_w), pH, and temperature on the lag phases, growth, and aflatoxin production of 3 A. flavus strains (CQ7, CQ8, and CG103) on a cheese-based medium. The results showed that the behavior of A. flavus strains was influenced by pH, a_w, and temperature conditions. The CQ7 strain showed the maximum growth at pH 5.5, 0.99 a_w, and 25°C, whereas for CQ8 and CQ103 strains, no differences were obtained at pH 5.5 and 6.0. In general, low pH, a_w, and temperature values increased the latency times and decreased the growth rate and colony diameter, although a_w and temperature were the most limiting factors. Maximum aflatoxin production on the cheese-based medium occurred at pH 5.0, 0.95 a_w, and 25 or 30°C, depending on the strain. This study shows the effect of pH, a_w, and temperature factors on growth and aflatoxin production of 3 aflatoxigenic A. flavus strains on a cheese-based medium. The findings may help to design control strategies during the cheesemaking process and storage, to prevent and avoid aflatoxin contamination by aflatoxigenic molds.     

Modeling of germination and growth of ochratoxigenic isolates of Aspergillus ochraceus as affected by water activity and temperature on a barley-based medium

Aspergillus ochraceus is an ochratoxin producing fungus that can be found on stored cereal grains such as barley. The objective of this study was to determine the effects of water activity (a_w, 0.75–0.99) and temperature (10–30°C) on germination and growth on barley extract agar medium (BMEA) of three isolates of Aspergillus ochraceus. The three isolates showed an optimal a_w for germination and growth of 0.99–0.95 at 20–30°C, with a marked increase of the lag phases and decrease of germination and growth rates at the marginal levels of a_w and temperature assayed. Minimum level of a_w for germination was 0.80 and 0.85 for growth. Data were then modeled by an MLR regression and response surface models were obtained. These models may allow a rough prediction of germination/growth as a function of the storage temperature and moisture content of barley grains.     

Comparison of dry matter losses and aflatoxin B1 contamination of paddy and brown rice stored naturally or after inoculation with Aspergillus flavus at different environmental conditions

The objective of this study was to compare the effect of different storage moisture conditions (0.70, 0.85, 0.90 and 0.95 water activity, a_w) and temperatures (20, 25, 30 °C) on (a) respiration rates (R) and dry matter loss (DML) of paddy and brown rice and (b) quantify aflatoxin B₁ (AFB₁) production by isolates of Aspergillus flavus from the rice samples and (c) inoculation of both rice types with A. flavus under these storage conditions on R, DML and AFB₁ contamination. There was an increase in temporal CO₂ production with wetter and warmer conditions in naturally contaminated rice. Higher R and consequently, % DML, were generally found in the brown rice (21%) while in paddy rice this was only up to 3.5% DML. From both rice types, 15 (83.3%) of 18 A. flavus isolates produced detectable levels of AFB₁ in a range 2.5–1979.6 μg/kg. There was an increase in DML in both rice types inoculated with A. flavus as temperature and a_w were increased. Interestingly very little AFB₁ was detected in paddy rice, but significant contamination occurred in the brown rice. The %DML in the control and A. flavus inoculated rice increased with temperature and a_w at both 25 and 30 °C from 1-2% to 15–20% DML at 30 °C and 0.95 a_w. All the inoculated rice samples had AFB₁ levels above the EU legislative limits for contamination in other temperate cereals and products derived from cereals (=2 μg/kg). Even samples with % DML as low as 0.2% had AFB₁ contamination levels twice the limits for other cereals. These results suggest that the mycotoxin contamination risk in staple commodities like rice, is influenced by whether the rice is processed or not, and that measurement of R rates can be used to predict the relative risk of AFB₁ contamination in such staple commodities.     

Development and application of a predictive model of Aspergillus candidus growth as a tool to improve shelf life of bakery products

Molds are responsible for spoilage of bakery products during storage. A modeling approach to predict the effect of water activity (a_w) and temperature on the appearance time of Aspergillus candidus was developed and validated on cakes. The gamma concept of Zwietering was adapted to model fungal growth, taking into account the impact of temperature and a_w. We hypothesized that the same model could be used to calculate the time for mycelium to become visible (t_v), by substituting the matrix parameter by t_v. Cardinal values of A. candidus were determined on potato dextrose agar, and predicted t_v were further validated by challenge-tests run on 51 pastries. Taking into account the a_w dynamics recorded in pastries during reasonable conditions of storage, high correlation was shown between predicted and observed t_v when the a_w at equilibrium (after 14 days of storage) was used for modeling (A_f = 1.072, B_f = 0.979). Validation studies on industrial cakes confirmed the experimental results and demonstrated the suitability of the model to predict t_v in food as a function of a_w and temperature.     

Effects of oilseed substrates (ground nyjer and flax seeds) on the growth and Ochratoxin A production by Aspergillus carbonarius

Aspergillus carbonarius is one of the major Ochratoxin A (OTA) producing fungus. Nyjer and flax seeds are important oilseeds that are used for both human and animal consumption, but they are highly susceptible to fungal growth and mycotoxin contamination. The objectives of this study were to determine the growth and OTA production by A. carbonarius on ground nyjer and flax seeds with water activity levels ranging from 0.82 to 0.98 a_w at three incubation temperatures (20, 30, 37°C). It was found that A. carbonarius was not able to grow on the two types of oilseeds with 0.82 or 0.86 a_w. Also, the fungus was not able to grow on flax seeds with high water activity (0.98 a_w). The OTA was only detected on flax seed samples with 0.94 a_w at 20°C. On nyjer seeds, the highest concentration of OTA (271 μg/kg) was detected from samples with 0.98 a_w incubated at 20°C for 5 days, while on flax seeds the highest OTA (146 μg/kg) was found on the seed samples with 0.94 a_w incubated at 20°C for 15 days. Linear regression models also indicated that 0.98 a_w was optimal for both fungal growth and OTA production on nyjer seeds. Overall, ground nyjer seed is better than flax seed to support growth and OTA production by A. carbonarius.     

A Predictive Model for the Growth/No Growth Boundary of Zygosaccharomyces bailii at 7?°C and Conditions Mimicking Acidified Sauces

The spoilage potential of Zygosaccharomyces bailii has been widely recognized within the food industry. However, few data are available on the growth characteristics of this yeast at low temperature and in the absence of chemical preservatives. In this study, the growth/no growth boundary of Z. bailii was defined at refrigeration temperature (7?°C) and at conditions relevant to high-sugar, low-pH foods (such as ketchup and salad dressing), i.e. pH?3.0?C5.0 (five levels), a _w 0.93?C0.97 (five levels) and acetic acid concentration 0?C2.5% (v/v; six levels). Yeast growth was followed during 90?days by optical density measurements, and logistic regression models were used to describe the data. Acetic acid had a significant effect on the relation between a _w and NaCl concentration and this interaction had important consequences for the model development. When data were modelled as a function of a _w or NaCl concentration, a stimulatory effect by acetic acid was observed. In contrast, as a function of (toxic) Na⁺ ions, no evidence was found of such a phenomenon. These results indicate that in cases where the relationship between a _w and solute concentration is not straightforward such as in the presence of acid preservatives, one must be critical towards the interpretation of data and correspondingly, the development of predictive models. On a practical note, the developed models, especially the one incorporating Na⁺ ions, may be used (1) to assess the stability of shelf-stable acidified foods stored under chilled conditions after opening or (2) to formulate new additive-free products intended for storage at 7?°C.     

Modeling Penicillium Expansum Growth Response to Thyme Essential oil at Selected Water Activities and pH Values Using Surface Response Methodology

Our objective was to evaluate, using a full factorial design, the effects of selected water activities (0.990, 0.945, or 0.900), pHs (5, 4, or 3), and thyme essential concentration (TEO, 0, 25, 50, or 100ppm) oil on Penicillium expansum lag time (λ) and radial growth rate (μ_m) obtained by modeling mold response using Gompertz equation, and corresponding polynomial quadratic models. Potato-dextrose agar formulated with every studied factor combination was inoculated with 10³ spores/ml, and incubated at 25°C up to 30 days. Mold colony diameter was periodically measured during incubation and adjusted with Gompertz equation to determine λ and μ_m. Decreasing a_w and pH, and increasing TEO concentration decreased μ_m and increased λ. At low a_w and pH, the increase in TEO concentration had a dramatic effect on P. expansum response since 25ppm of TEO inhibited its growth for 30 days at 25°C. Gompertz parameters exhibited that P. expansum was sensitive to the evaluated combined factors, allowing us to construct a secondary predictive growth model. TEO in combination with a_w and pH reduction effectively inhibited P. expansum growth.     

Effect of Water Activity aw of Milk on acid Production by Streptococcus thermophilus and Lactobacillus bulgaricus

Rate of acid production by Lactobacillus bulgaricus ATCC 11842 and Streptococcus thermophilus ATCC 19258 was measured at temperatures between 30°C and 50°C in milks which had been adjusted from 0.992 to 0.943 a_w with glycerol. Data were fitted with a nonlinear mathematical model of the Arrhenius type. Decrease of a_w inhibited acid production by both organisms. Range of temperatures, within which the rate of acid production was higher than 50% of the optimal rate, was also narrowed with both species. Optimal temperatures of acid production were unaffected by changes of a_w. Minimum a_w for acid production was always lower when a_w of milk was adjusted with glucose or sucrose than with glycerol.     

Modeling the effect of natamycin,pine-resin and environmental factors on the growth and OTA production by Aspergillus carbonarius using response surface methodology

The effect of two antifungal compounds (natamycin, pine-resin), temperature and water activity, on the growth rate, lag phase duration and Ochratoxin A (OTA) production by three Aspergillus carbonarius isolates (Ac-28, Ac-29, and Ac-33), was studied by means of Response Surface Methodology (RSM) based on a Central Composite Design (CCD). Two different experimental designs were performed as a function of temperature (16.6–33.4 °C), water activity (0.90–0.97 a_w), natamycin (0–1000 ng ml^− 1) or pine-resin (0–2.61%, w/v) on a Synthetic Grape-juice Medium (SGM). OTA production was analyzed after 5, 10 and 15 days of incubation. A second-order polynomial model was fitted to each response parameter to assess the growth and OTA potential of all fungal isolates. Results showed that natamycin, a_w and temperature had significant effects on the lag phase duration of all isolates, as well as on OTA accumulation after 10 days of incubation for Ac-29 and 15 days for Ac-28 and Ac-33 isolates. The same results were obtained for OTA production after treatment with pine-resin. However, fungal growth rates were not statistically significant in both experiments, with the exception of Ac-29 and Ac-33 after treatment with pine-resin. Overall, high natamycin concentrations (800 and 1000 ng ml^− 1) delayed fungal growth depending on the environmental factors assayed. Moreover, treatment with pine-resin at 16.6 °C/0.94 a_w/1.1% w/v, as well as at 25 °C/0.90 a_w/1.1% w/v, completely inhibited fungal growth up to 15 days of incubation.     

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.     

Is intraspecific variability of growth and mycotoxin production dependent on environmental conditions? A study with Aspergillus carbonarius isolates

The aim of this study was to assess the impact of suboptimal environmental conditions on the intraspecific variability of A. carbonarius growth and OTA production using thirty isolates of A. carbonarius.Three a_w/temperature conditions were tested, one optimal (0.98a_w/25 °C) and two suboptimal: 0.90a_w/25 °C and 0.98a_w/37 °C as suboptimal water activity and temperature, respectively, which might take place through over ripening and dehydration of grapes. For each condition, 12 Petri dishes were inoculated, and colony growth and OTA production were measured over time.ANOVA revealed significant differences among μ and λ within the 30 assayed isolates. Coefficients of variation (CV%) revealed a wider dispersion of growth rates at 0.90a_w/25 °C compared to 0.98a_w/25 °C, and a more than 4-fold higher CV at 0.98a_w/37 °C compared to 0.98a_w/25 °C. However, dispersion of lag phases was similar at 0.98a_w/25 °C and 0.90a_w/25 °C and wider at 0.98a_w/37 °C.There were significant differences (p < 0.05) among OTA levels (ng/mm²) for the different conditions, values being lower under marginal conditions, and particularly at 0.98a_w/37 °C. Coefficients of variation (CV%) revealed a wider dispersion of OTA production at 0.90a_w/25 °C compared to 0.98a_w/25 °C, while CV at 0.98a_w/37 °C was similar to that at 0.98a_w/25 °C.In order to address the strain variability in growth initiation and prove the well-established notion of reducing OTA in foods by preventing fungal growth, a greater number of strains should be included when developing models for conditions that are suboptimal both for a_w for OTA production and temperature levels for growth.