Modelling of effects of water activity and temperature on germination and growth of ochratoxigenic isolates of Aspergillus ochraceus on a green coffee-based medium

Influence of water activity (0.75-0.99 a(w)) and temperature (10, 20 and 30 degrees C) on germination and mycelial growth on green coffee extract agar medium of three ochratoxigenic isolates of Aspergillus ochraceus was studied. Optimal conditions for germination and growth were observed at 0.95-0.99 a(w) and 20-30 degrees C for the three isolates. Minimum a(w) level for germination was 0.80, and 0.85 for mycelial growth. At marginal a(w) and temperature levels assayed, the lag phases prior to germination increased and the growth rates showed a significant decrease in comparison with the optimal conditions. Data were modelled by a multiple linear regression (MLR) and response surface models were obtained. Germination and growth of A. ochraceus in green coffee beans could be prevented or at least inhibited to some extent by minimising the time that coffee beans are exposed to temperature and humidity conditions near to the optimum during processing and storage. This could be an empirical approach to predict the effects of water activity and temperature conditions on the development of ochratoxigenic isolates of A. ochraceus during handling and storage of green coffee.     

Influence of water activity and temperature on conidial germination and mycelial growth of ochratoxigenic isolates of Aspergillus ochraceus on grape juice synthetic medium. Predictive models

The first stages in the development of Aspergillus ochraceus, an ochratoxin A‐producing fungus that infects grapes and may grow on them, have been studied on a synthetic nutrient medium similar to grape in composition. Spore germination and mycelial growth have been tested over a water activity (a_w) and temperature range which could approximate to the real conditions of fungal development on grapes. Optimal germination and growth were observed at 30 °C for all three isolates tested. Maximal germination rates were detected at 0.96–0.99 a_w at 20 °C, while at 10 and 30 °C the germination rates were significantly higher at 0.99 a_w. Although this abiotic factor (a_w) had no significant influence on mycelial growth, growth rates obtained at 0.98 a_w were slight higher than those at other a_w levels. Predictive models for the lag phase before spore germination as a function of water activity and temperature have been obtained by polynomial multiple linear regression, and the resulting response surface models have been plotted. Copyright © 2005 Society of Chemical Industry     

Impact of relative humidity and temperature on visible fungal growth and OTA production of ochratoxigenic Aspergillus ochraceus isolates on grapes

Aspergillus ochraceus is an ochratoxin A (OTA) producer mould found in grapes and this may contribute to OTA contamination in wines and grape juices. The influence of relative humidity (R.H.; 80, 90 and 100%) and temperature (10, 20 and 30 °C) on visible mould growth on grapes and OTA accumulation after 14 days of incubation by this fungal species has been studied using a full factorial design with three replicates.The two abiotic factors and their interaction (R.H.×temperature) affected significantly the A. ochraceus growth in berries, which was maximum at 90–100% R.H. levels. With regard to the optimum temperature level, it occurred at 30 °C at 80 and 90% R.H., whereas no significant differences were detected at 20 and 30 °C when R.H. was 100%.OTA production by A. ochraceus on grapes was not significantly modified by the assayed levels of temperature and R.H, with a mean value of 3.53 ng g⁻¹.Predictive models of percentage of grapes with visible growth of A. ochraceus isolates under different relative humidity and temperature are presented.     

Effects of water activity and temperature on germination and growth profiles of ochratoxigenic Penicillium verrucosum isolates on barley meal extract agar

The aim of this work was to study the combined effects of water activity (aw, 0.75-0.99), temperature (10-30 degrees C) and isolate on spore germination and mycelial growth of ochratoxigenic Penicillium verrucosum isolates on barley meal extract agar (BMEA). Lag phases prior to germination (h), germination rates (h(-1)) and growth rates (mm day(-1)) were obtained at each set of conditions and for all the isolates. Analysis of variance showed a significant influence of the abiotic factors assayed on fungal development. Minimum aw levels found for germination and mycelial growth were 0.80 and 0.85, respectively, while maximum germination and growth rates were obtained at 0.95-0.99 aw level. Although maximum germination was obtained at 20 degrees C, significant differences were not found between growth rates at 20 and 30 degrees C. Data were modelled by MLR regressions and response surface models were obtained. Spore germination and mycelial growth of ochratoxigenic P. verrucosum isolates are significantly affected by water activity and temperature. Moreover, different isolates may have slightly different response to environmental factors. Predictive models obtained may allow a rough prediction of germination/growth as a function of the storage temperature and moisture content of barley grains.     

Effect of water activity and temperature on mycelial growth and ochratoxin A production by isolates of Aspergillus ochraceus on irradiated green coffee beans

Aspergillus ochraceus as a fungal contaminant and ochratoxin A (OTA) producer plays an important role in coffee quality. Temperature and water activity (a(w)) significantly influence mycelial growth and OTA production by isolates of A. ochraceus on green coffee beans. Maximum mycelial growth was found at 30 degrees C and 0.95 to 0.99 a(w). A marked decrease in growth rate was observed when temperature and a(w) were reduced. At 0.80 a(w), mycelial growth occurred only at 30 and 20 degrees C for one isolate. Maximum OTA production was found at 20 degrees C and 0.99 a(w). At 10 degrees C, OTA was not produced, regardless of a(w). Similarly, no OTA was detected at 0.80 a(w). OTA production ranged from the limit of detection (40 ng g(-1) of green coffee) to 17,000 ng g(-1) of green coffee. Significant intraspecific differences in mycelial growth and OTA production were found. Primary data for lag phases prior to mycelial growth under the influence of temperature and a(w) were modelled by multiple linear regression, and the response surface plots were obtained.     

Prediction of fungal growth and ochratoxin A production by Aspergillus ochraceus on irradiated barley grain as influenced by temperature and water activity

Ochratoxin A (OTA) is a secondary metabolite of Aspergillus and Penicillium species, including Aspergillus ochraceus, a species that can be found in stored cereal grains such as barley. The objective of this study was to determine the effects of water activity (a(w), 0.80-0.99), temperature (10, 20, 30 degrees C), and A. ochraceus isolate differences on radial growth and OTA production in irradiated barley grains. The three isolates showed optimal conditions for growth and ochratoxin A production at 0.99 a(w) and 30 degrees C, with a marked decrease of growth rates and OTA production at the lowest levels of a(w) and temperature assayed. The minimum a(w) level for growth, observed in this study, was 0.85 and 0.90 a(w) for OTA production. Significant differences among the isolates were found. Lag phases prior to fungal growth and OTA production values were modelled by multiple linear regression and response surface models. These models could provide an approximate prediction of growth and OTA production.     

Impact of non-selective fungicides on the growth and production of ochratoxin A by Aspergillus ochraceus and A. carbonarius in barley-based medium

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, copper oxychloride, and sulfur on the growth and capability for producing ochratoxin A (OTA) of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based medium. Lag phases and growth rates were determined for each fungicide at different doses, at 15°C and 25°C and at 0.97 a(w). Mancozeb at 40 mg l(-1 )inhibited fungal growth and provided lag phases >24 days at 10-20 mg l(-1) and 15°C. OTA was observed only at 25°C and doses <10 mg l(-1). At 15°C, copper oxychloride proved inhibitory at 800 mg l(-1), while at 25°C growth was not delayed and only high doses decreased OTA levels. Sulfur was inhibitory or provided large lag phases at 5-8 g l(-1) (at 15°C) while at 25°C growth took place even at 8 g l(-1), although OTA levels were low or undetectable. The antifungal activity decreased in the order mancozeb > copper oxychloride > sulfur, and was lower at 25°C than at 15°C. OTA accumulation was affected by the type of fungicide, dose, temperature and time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and OTA production in barley-based medium is assessed for the first time.     

Impact of non-selective fungicides on the growth and production of ochratoxin A by Aspergillus ochraceus and A. carbonarius in barley-based medium

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, copper oxychloride, and sulfur on the growth and capability for producing ochratoxin A (OTA) of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based medium. Lag phases and growth rates were determined for each fungicide at different doses, at 15°C and 25°C and at 0.97?a_w . Mancozeb at 40?mg?l^?1 inhibited fungal growth and provided lag phases >24 days at 10–20?mg?l^?1 and 15°C. OTA was observed only at 25°C and doses <10?mg?l^?1. At 15°C, copper oxychloride proved inhibitory at 800?mg?l^?1, while at 25°C growth was not delayed and only high doses decreased OTA levels. Sulfur was inhibitory or provided large lag phases at 5–8?g?l^?1 (at 15°C) while at 25°C growth took place even at 8?g?l^?1, although OTA levels were low or undetectable. The antifungal activity decreased in the order mancozeb?>?copper oxychloride?>?sulfur, and was lower at 25°C than at 15°C. OTA accumulation was affected by the type of fungicide, dose, temperature and time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and OTA production in barley-based medium is assessed for the first time.     

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.     

Ecophysiology of ochratoxigenic Aspergillus ochraceus and Penicillium verrucosum isolates. Predictive models for fungal spoilage prevention - a review

Ochratoxin A (OTA) is a secondary metabolite produced by several species of Aspergillus and Penicillium; among them Aspergillus ochraceus and Penicillium verrucosum are two ochratoxigenic species capable of growing in different climates and thus contamination of food crops with OTA can occur worldwide. OTA can be found in a wide range of foods such as cereals, coffee, cocoa, spices, beer, wine, dried vine fruit, grapes and meat products. OTA is toxic to animals, it presents neurotoxic, immunotoxic and nephrotoxic effects. It has been implicated in a human kidney disorder known as Balkan Endemic Nephropathy. This review focuses on the ecophysiology of ochratoxin-producing Aspergillus ochraceus and Penicillium verrucosum, the effect of environmental factors on their germination, mycelial growth, and OTA production. Knowledge of environmental conditions required for sucessive stages of fungal development represent the first step towards preventing mycotoxin formation. Predictive models for different stages of fungal development are presented, which allow prediction of the time before spoilage as a function of the abiotic factors. Finally, the implications of these studies in management of barley, coffee and grapes are described. This can help to identify the critical control points in their production, storage and distribution processes.     

Effect of water activity and temperature on growth of ochratoxigenic strains of Aspergillus carbonarius isolated from Argentinean dried vine fruits

Aspergillus carbonarius is an ochratoxin A (OTA) producing fungus, predominantly responsible for the production of this mycotoxin in grapes, wine and dried vine fruits. The objective of this study was to determine the in vitro effects of water activity (a(w), 0.80-0.95) and temperature (15-35 degrees C) on lag phase extension and radial growth rate of a cocktail inoculum of four strains of A. carbonarius. The maximum growth rate was observed at 0.95 a(w) and 30 degrees C (17.46 mm day(-1)). In general, growth rates increased with the increment of a(w). No growth was observed at a(w) below 0.85. For all a(w) levels tested the highest growth rate was detected at 30 degrees C. At 15 degrees C growth only occurred at the higher a(w) levels evaluated (0.925 and 0.95) at a growth rate of 3.82 and 5.57 mm day(-1) respectively. The shortest lag phase (0.26 days) was found at 0.925 a(w) and 35 degrees C. At marginal conditions of a(w) and temperature the lag phases increased, being the highest registered at 20 degrees C and 0.89 a(w) (33.7 days). The pattern of effects of environmental factors on growth was similar among Argentinean A. carbonarius strains and those from several European countries, Israel and Australia.     

Effect of temperature and water activity on growth and ochratoxin A production by Australian Aspergillus carbonarius and A. niger isolates on a simulated grape juice medium

The effect of water activity (0.92, 0.95, 0.965 and 0.98) and temperature (15 °C, 25 °C, 30 °C and 35 °C) on growth rate and ochratoxin A (OA) production by five strains of Aspergillus carbonarius and two strains of A. niger isolated from Australian vineyards was characterised on a synthetic grape juice medium. Maximum growth for A. carbonarius occurred at ca 0.965 a_w and 30 °C, and for A. niger, at ca 0.98 a_w and 35 °C. The optimum temperature for OA production was 15 °C and little was produced above 25 °C. The optimum a_w for toxin production was 0.95–0.98 for A. carbonarius and 0.95 for A. niger. Toxin was produced in young colonies after and, typically, did not continue to accumulate the entire surface area of the plate was colonised. Rather, the amount decreased as colonies aged. Trends for growth and OA production were similar among Australian isolates and those from European grapes, as reported in the literature.     

Minimal water activity for growth of Listeria monocytogenes as affected by solute and temperature.

Two strains of Listeria monocytogenes, Scott A (serotype 4b) and Brie 1 (serotype 1b) were characterized in terms of minimum aw requirements for growth at 4 and 30 degrees C, using NaCl, glycerol and sucrose as test solutes. Both strains grew well at 30 degrees C in glycerol-supplemented tryptic soy broth (TSB), but not in NaCl- and sucrose-supplemented TSB at aw 0.90. Sucrose was more inhibitory than NaCl and glycerol. The Brie 1 strain was less tolerant to sucrose compared to the strain Scott A at both 4 and 30 degrees C. The effects of all three solutes were magnified at 4 degrees C, where tolerance to low aw was markedly less than at 30 degrees C. Results confirm that the type of solute as well as the osmotic conditions created by the solute affects the ability of Listeria monocytogenes to grow at 4 and 30 degrees C.     

Comparative study of toxigenic potential of Aspergillus flavus and Aspergillus niger isolated from Barley as affected by temperature,water activity and carbon source

The objective of the present work was to evaluate the ability of two mycotoxigenic species Aspergillus flavus and Aspergillus niger, isolated from barley, to produce aflatoxin B1 (AFB1) and ochratoxin A (OTA), respectively, as affected by nutritional and environmental factors. Six carbon sources (D-fructose, D-glucose, D-galactose, lactose, sucrose and starch) and different water activities (0.90, 0.95 and 0.98), temperature (20 and 28 °C) and incubation time (5 and 10 days) were tested. The results showed that optimal conditions for growth on Barley Meal Extract Agar (BMEA) medium were 28 °C and 0.95 a_w for A. niger strain and 28 °C and 0.98 a_w for A. flavus strain. Optimal conditions for OTA and AFB1 production were largely different for the two tested strains. A. niger had an optimal OTA production at 0.98 a_w and 20 °C after 10 days of incubation while A. flavus had an optimal AFB1 production at 0.95–0.98 a_w and 28 °C after 5 days of incubation. These results indicates that A. flavus has a higher optimum temperature for mycotoxin synthesis than A. niger and takes greater advantage of drier conditions for maximum AFB1 production. In the current study, both OTA and AFB1 production of A. niger and A. flavus were highly influenced by carbon sources. The sugar that provided the highest toxin levels in the cultures of the two species was sucrose with the lowest levels given by starch. OTA production by A. niger was also highly induced by fructose as carbon sources, while, AFB1 production by A. flavus was favored by glucose. Globally, our results showed, significantly different optimal conditions for production of AFB1 and OTA, respectively, by A. flavus and A. niger.     

Modelling the effect of temperature and water activity of Aspergillus flavus isolates from corn

The aim of this study was to model the effects of temperature (10-40 °C) and a(w) (0.80-0.98), in two media (Czapek yeast agar: CYA; corn extract medium: CEM) on the growth rates and growth boundaries (growth-no growth interface) of three strains of A. flavus isolated from corn in Argentina. Both kinetic and probability models were applied to colony growth data. The growth rates obtained in CYA were significantly (p<0.05) greater than those obtained in CEM medium. No significant differences (p<0.05) were observed among the three isolates. The growth rate data showed a good fit to the Rosso cardinal models combined with the gamma-concept with R(2)=0.98-0.99 and RMSE=0.60-0.78, depending on media and isolates. The probability model allowed prediction of safe storage (p of growth <0.01) for one month for moist maize (e.g. 0.90 a(w)) provided temperature is under 15 °C, or for dry maize (e.g. 0.80 a(w)) provided temperature is under 27 °C. Storage at <0.77 a(w) would be safe regardless of the storage temperature. Probability models allow evaluation of the risk of fungal contamination in the process of storage, so the results obtained in this study may be useful for application in systems of food safety management.     

Impact of water activity and temperature on growth and ochratoxin A production of two Aspergillus carbonarius isolates from wine grapes in Greece

The aim of this study was to determine the effects of water activity (a(w); 0.85 to 0.98) and temperature (10 to 40 degrees C) on the radial growth rate and ochratoxin A (OA) production of two Aspergillus carbonarius isolates in vitro. The isolates were obtained from wine grapes cultivated in Greece, and the trial was conducted with a synthetic grape juice medium similar in composition to grapes between veraison (beginning of color change) and ripeness. Fungal growth and OA production data were collected for 55 days. Response surface curves and cardinal values for a(w) and temperature were obtained using multiple regression analysis. The lag phase lasted from less than 1 to 10 days. Both isolates grew optimally at 30 to 35 degrees C and 0.96 a(w), but maximum OA production occurred under suboptimal growth conditions (15 to 20 degrees C and 0.93 to 0.96 a(w)). Growth also was observed at 0.85 a(w) and 25 degrees C, however at this same a(w) the fungus failed to produce mycelium at any other temperatures tested. The isolates produced OA at 15 to 30 degrees C and 0.90 to 0.98 a(w). Maximum OA production was detected after 25 days of incubation at 20 degrees C and 0.96 a(w) and was 3.14 and 2.67 microg g(-1), respectively, for the two strains. The isolated strains used in this study were more xerotolerant than others from the Mediterranean basin. These data will allow producers to identify and thus monitor critical environmental conditions effectively in wine grapes. These data also increase the knowledge base concerning the ability of A. carbonarius to grow and produce toxin under different ecological conditions and can contribute to the development of secondary models for the prediction and risk assessment of OA in wine production.     

SEM study of water activity and temperature effects on the initial growth of Aspergillus ochraceus,Alternaria alternata and Fusarium verticillioides on maize grain. Scanning electron microscopy

A scanning electron microscopy (SEM) study of the effect of water activity (a(w)), temperature and fungal interactions on the very early phases of Aspergillus ochraceus, Alternaria alternata and Fusarium verticillioides development on maize grains was carried out. Germination and growth of individual fungal strains were assayed at 0.92, 0.95 and 0.98 a(w), and 20 and 30 degrees C. Hyphal lengths were measured on micrographs taken by SEM at different periods of incubation. A. alternata had the highest linear growth at 0.98 a(w), and was the only species able to grow under all conditions tested, whereas A. ochraceus was not able to germinate at 0.92 a(w) at any temperature assayed. F. verticillioides demonstrated a different behaviour depending on growth temperature. Fungal interactions were studied at 0.95a(w) and 30 degrees C. A. ochraceus germination was inhibited when it was co-inoculated with one or two of the other species. A. alternata showed an increased growth rate when growing together with the other fungi, whereas growth of F. verticillioides was significantly inhibited when paired with A. ochraceus.     

Non-specificity of nutritional substrate for ochratoxin A production by isolates of Aspergillus ochraceus

Aspergillus ochraceus is an important contaminant of diverse substrates, such as cereals, coffee, grapes and derivates. This fungus produce a nephrotoxic metabolite, ochratoxin A (OTA), whose presence on food and feeds may be an important risk for animal and human health. The aim of this work was to evaluate the significance of the origin of A. ochraceus isolates on their OTA production patterns on different substrates (yeast extract sucrose (YES) broth, irradiated barley grains, irradiated green coffee beans and sterilized grapes) and under different environmental conditions. Results did not show a significant influence of the isolation source on OTA-production profiles by A. ochraceus isolates on several substrates, since the isolates which produced the highest OTA amounts in vitro (YES medium) were also the isolates with the highest OTA yields on the other substrates. Abiotic factors assayed (water activity, temperature and substrate) affected significantly OTA productions by A. ochraceus. Maximum OTA amounts were detected at 25 degrees C and 0.98 a(w) on all substrates tested. The highest OTA accumulations found on the different substrates were: green coffee beans (> 2 mg g(-1)), barley grains (approximately 1 mg g(-1)), YES medium (13.9 microg ml(-1)) and grape (approximately 3 ng g(-1)).     

Predicting microbial growth: growth responses of salmonellae in a laboratory medium as affected by pH, sodium chloride and storage temperature

The growth responses of salmonellae (mixed inoculum of Salmonella thompson, S. stanley and S. infantis) as affected by NaCl concentration, pH level and storage temperature were studied in laboratory medium. Growth curves were obtained at 5 concentrations of NaCl (0.5-4.5%, w/v), 5 pH levels (5.6-6.8) and 5 storage temperatures (10-30 degrees C). Sigmoid curves (Gompertz form) were fitted to the data and the curve parameters used to produce a polynomial model from which predicted growth curves could be generated for any combination of NaCl, pH and storage temperature within the limits studied. From those growth curves values for growth rate, generation time, lag time and other values such as time to a 1000-fold increase in numbers were derived. Such a model offers a cost-effective approach to understanding the microbial growth response in foods, and forms a data-base against which other controlling factors could be evaluated. Some problems of fitting curves to microbial growth data and of modelling such data are discussed.     

Modeling the germination kinetics of clostridium botulinum 56A spores as affected by temperature, pH, and sodium chloride

The germination kinetics of proteolytic Clostridium botulinum 56A spores were modeled as a function of temperature (15, 22, 30 degrees C), pH (5.5, 6.0, 6.5), and sodium chloride (0.5, 2.0, 4.0%). Germination in brain heart infusion (BHI) broth was followed with phase-contrast microscopy. Data collected were used to develop the mathematical models. The germination kinetics expressed as cumulated fraction of germinated spores over time at each environmental condition were best described by an exponential distribution. Quadratic polynomial models were developed by regression analysis to describe the exponential parameter (time to 63% germination) (r2 = 0.982) and the germination extent (r2 = 0.867) as a function of temperature, pH, and sodium chloride. Validation experiments in BHI broth (pH: 5.75, 6.25; NaCl: 1.0, 3.0%; temperature: 18, 26 degrees C) confirmed that the model's predictions were within an acceptable range compared to the experimental results and were fail-safe in most cases.