Vanillin and pH Synergistic Effects on Mold Growth

The combined effects of pH (3.0–4.0) and vanillin concentration (500–1,000 ppm) on the growth of Aspergillus flavus, A. niger, A. ochraceus and A. parasiticus were evaluated in potato-dextrose agar adjusted to a water activity 0.98. Mold germination time and radial growth rates (RGR) were significantly affected by the variables (p<0.05). For equal concentration of the antimicrobial the reduction in pH had important effects lowering the RGR. The lag time increased as vanillin concentration increased and pH decreased. The inverse lag times of each mold could be described by second order polynomials.     

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.     

OPTIMIZATION OF NUTRIENTS AND CULTIVATION CONDITIONS IN GLUCOAMYLASE PRODUCTION

Initial adjustment of pH of growth medium in the range of 3.0–5.0 was found to be necessary for optimal production of glucoamylase by a newly isolated strain of Aspergillus niger. Optimum temperature of incubation under stationary conditions was found to be 26–28° C, although incubation at higher temperatures viz. 30 and 37° C led to faster fungal growth rate and increased biomass. In complex medium, the optimum glucose and peptone concentrations were 2 and 3% respectively, and the presence of trace elements was found to be essential for maximal enzyme yield.     

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.     

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.     

Influence of temperature on growth rate and lag phase of fungi isolated from Argentine corn

The influence of temperature on the growth of nine strains of fungi belonging to the genera Eurotium, Aspergillus, Penicillium and Fusarium has been investigated for the temperature range 15–35° C. The lag phase and the growth rate were evaluated by using a laboratory medium. The maximum growth rate for E. repens, A. wentii and P. chrysogenum was observed at about 25° C, for P. citrinum near 30° C and for F. semitectum and F. moniliforme between 20 and 25° C. The growth rate of A. niger, A flavus and A. parasiticus increased with increasing temperatures in the range studied. For all strains studied it appeared that the higher the growth rate the lower the lag phase was.     

Induction of a lag phase by chiller temperatures in Escherichia coli growing in broth or on pork

When log phase cultures of Escherichia coli in brain–heart infusion (BHI) were cooled from 12°C to temperatures below 7°C (the minimum required for growth), the optical densities of cultures increased at declining rates for up to 6 days during incubation at temperatures between 6 and 3°C inclusive, but did not increase at temperatures ≤2°C. However, the numbers of E. coli recovered from cultures on selective or non-selective agars were similar at all times of incubation up to 8 days at temperatures below the minimum for growth. From optical density measurements it appeared that when log phase cultures were cooled to 2°C, then returned to 15°C, a lag developed with time at 2°C to reach a maximum of about 1 h after about 4 h. When cultures were returned to 12°C after times at 2°C between 0·5 and 8 days, the time during which optical densities did not increase was constant at about 2 h, but the initial rate of optical density increase declined with time at 2°C. On pork fat tissue the lag time, determined by increases in the numbers of E. coli cfu on tissues inoculated with cells returned from incubation at 2°C for 16 h to a growth permitting temperature between 7 and 30°C, was longer than the lag time determined from optical density measurements of broth cultures subjected to the same temperature regime. Lag times for E. coli adapted to and growing on normal pH pork lean tissue were longer than for E. coli on fat tissue, while unadapted E. coli did not initiate growth on lean tissue, after incubation at 2°C, at 12°C or lower temperatures. The observations indicate that lag phase development in log phase E. coli subjected to chiller temperatures is complex, and that prediction of lag resolution in log phase cells exposed to temperatures that fluctuate around the minimum for growth will require modeling of lag induction as well as lag resolution.     

Increased Aflatoxin Production by Aspergillus parasiticus Under Conditions of Cycling Temperatures

The effects of cycling temperatures (5°C for 12 hr and 25°C for 12 hr) on aflatoxin production by Aspergillus parasiticus NRRL 2999 in yeast extract sucrose (YES) medium were studied. Cycling temperatures, after preincubation at 25°C for various times, resulted in more aflatoxin B₁, G₁, and total aflatoxin production than did constant incubation at either 25°C, which is generally considered to be the optimum for aflatoxin production, or 15°C, which is the same total thermal input as the 5-25°C temperature cycling. With increased preincubation time at 25°C, toxin production increased and the lag phase of growth was shortened or not evident. Cultures that were preincubated at 25°C for 1, 2, and 3 days prior to onset of temperature cycling showed the greatest increase in maximum aflatoxin production over the 25°C and 15°C constant temperatures. Cultures that were not preincubated at 25°C but subjected to constantly fluctuating temperatures produced maximum amounts of aflatoxin equivalent to cultures incubated at a constant 25°C. The maximum aflatoxin production at all temperatures studied occurred during the late log phase of growth and at pH minimums. Aflatoxins were found in higher concentrations in the broth than the mycelia under temperature cycling conditions, at 15°C, and at 25°C during the first 21 days of incubation, whereas greater amounts of toxin were retained in mycelium at 25°C in the later incubation period (28-42 days).     

Characterisation of acid proteases from a fusant F76 and its progenitors Aspergillus oryzae HN3042 and Aspergillus niger CICC2377

The characteristics of a novel acid protease from a fusant F76 were comparatively evaluated with those from its progenitors Aspergillus oryzae HN3042 and A. niger CICC2377. The UV spectra of these three acid proteases were similar, but fluorescence spectra were different. The acid protease from F76 contained 7.1% α‐helix, 39.4% β‐sheet, 24.7% β‐turn and 32% aperiodic coil, unlike those from its progenitors. The acid protease from F76 was active in the temperature range of 35–55 °C with the optimum temperature of 40 °C and was stable in the pH range of 2.5–6.5 with the optimum pH of 3.5, while those values from A. oryzae HN3042 and A. niger CICC2377 were 45 °C, 4.0 and 40 °C, 3.5, respectively. The kinetic parameters of the acid protease from F76 were different from its progenitors and the Michaelis constant, maximum velocity, activation energy, and attenuation index were 0.96 mg mL^?1, 135.14 μmol min^?1 mg^?1, 64.11 kJ mol^?1 and 0.59, respectively.     

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.     

Water activity and temperature effects on growth of Aspergillus niger, A. awamori and A. carbonarius isolated from different substrates in Argentina

The objectives of this study were to determine the effect of water activity, temperature, and their interactions on a) mycelial growth rate and b) the lag phase prior to grow of seven isolates of Aspergillus section Nigri isolated from peanuts, maize kernels, dried grapes and coffee cherries from Argentina. Three Aspergillus niger, three A. awamori and one A. carbonarius isolates examined showed optimum a(W) level for growth at 0.97 with optimal temperature of 30 degrees C. for most of the isolates and 25 degrees C for only one (A. awamori RCP176). Minimal a(W) for growth was 0.85 at the highest temperature tested. Overall growth was reduced up to 50% at 0.93 a(W). Growth was also to a large extend inhibited at 0.85 a(W) for most isolates even after 21 days of incubation at temperatures lower than 30 degrees C. The analysis of variance of the effect of single (isolate, a(W) and temperature), two- and three-way interaction showed that all factors alone and all interactions were statistically significant (P<0.001) in relation to growth rates and lag phase for A. niger, A. awamori and A. carbonarius isolates. These data are relevant since these species are isolated in high frequency on numerous substrates for human and animal consumption in Argentina.     

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     

Influence of white light,near-UV irradiation and other environmental conditions on production of aflatoxin B1 by Aspergillus flavus and ochratoxin A by Aspergillus ochraceus

The effects of illumination, near-ultraviolet, incubation temperature pH and some minor elements on the growth rate and production of aflatoxin B₁ by A. flavus and ochratoxin A by A. ochraceus were investigated. Aflatoxin B₁ and ochratoxin A production was considerably higher in the light than in the dark. The greatest aflatoxin B₁ and ochratoxin A production was occurred after 11 days of fermentation with light- and dark-grown cultures at 25 °C. The mycelial dry weight was also greater in the light than in the dark for both A. flavus and A. ochraceus. Exposure of conidia to near-UV irradiation increased mycelial dry weight and mycotoxins by both fungi more than white light. The greatest aflatoxin B₁ and ochratoxin A was at 25 °C with UV-grown culture (24 h exposure) producing a mean of 400 and 260 μg/50 ml of medium, respectively. The maximum aflatoxin B₁ and ochratoxin A yield was obtained at pH 5.5 and with increasing the initial pH to near neutrality, both mycotoxins yield decreased. Iron, cupper and zinc were observed to stimulate aflatoxin B₁ and ochratoxin A production and enhanced the growth rate of both A. flavus and A. ochraceus.     

Aspergillus flavus dose-response curves to selected natural and synthetic antimicrobials

The effects of selected concentrations of antimicrobials from natural (vanillin, thymol, eugenol, carvacrol or citral) or synthetic (potassium sorbate or sodium benzoate) origin on Aspergillus flavus lag time inoculated in laboratory media formulated at water activity (a(w)) 0.99 and pH 4.5 or 3.5, were evaluated. Time to detect a colony with a diameter > 0.5 mm was determined. Mold response was modeled using the Fermi function. Antimicrobial minimal inhibitory concentration (MIC) was defined as the minimal required inhibiting mold growth for 2 months. Fermi function successfully captured A. flavus dose-response curves to the tested antimicrobials with a highly satisfactory fit. Fermi equation coefficients, Pc and k, were used to compare antimicrobials and assess the effect of pH. Important differences in Pc and k were observed among antimicrobials, being natural antimicrobials less pH dependent than synthetic antimicrobials. A large Pc value represents a small antimicrobial effect on A. flavus lag time; thus, high concentrations are needed to delay growth. A. flavus exhibited higher sensitivity to thymol, eugenol, carvacrol, potassium sorbate (at pH 3.5), and sodium benzoate (at pH 3.5) than to vanillin or citral. MICs varied from 200 ppm of sodium bcnzoate at pH 3.5 to 1800 ppm of citral at both evaluated pHs.     

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.     

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.     

Fungitoxic activity of Indian borage (Plectranthus amboinicus) volatiles

Indian borage (Plectranthus amboinicus) was investigated for antifungal activity through agar well diffusion assay. Indian borage oil (IBO) was found to be effective against various fungi tested, as it inhibited the radial growth of mycelia and exhibited broad fungitoxic properties against Aspergillus flavus, Aspergillus niger, Aspergillus ochraceus CFR 221, Aspergillus oryzae, Candida versatilis, Fusarium sp. GF-1019, Penicillium sp., and Saccharomyces cerevisiae. The effective concentration of IBO on the growth of A. ochraceus in yeast extract sucrose medium was determined. IBO completely inhibited ochratoxin (OTA) production by the toxigenic strain A. ochraceus at 500 ppm. Also, the application of IBO at 100 mg/g in food samples resulted in inhibition of the growth of A. ochraceus in food systems such as groundnut, maize and poultry feed and no detectable amount of OTA was found at a high moisture level of 30%, even after seven days. IBO has the potential for use as a botanical fungitoxicant against fungal attack in stored food commodities.     

Relationship between thiosulfinates and pink discoloration in onion extracts,as influenced by pH

The influence of immediate and delayed pH control, and added S-methyl-L-cysteine sulfoxide (MCSO) on the progress of pink discoloration in relation to the fate of thiosulfinates in extracts of yellow onion (Allium cepa) bulbs was evaluated over an incubation period of about 3 weeks at 21–24°C. Greatest discoloration occurred in extracts prepared with immediate pH control at 6.1, and least discoloration occurred at pH 3.2, over the pH range of 3.2–6.1. Initial thiosulfinate levels, in descending order of abundance, occurred at pH 6.1 > 5.0 > 4.2 > 3.2 for extracts subject to immediate pH control. However, over 21 days incubation, the lowest residual levels (ranging 20–60% of maximum levels) were observed for extracts adjusted to pH 3.2 and 6.1. Extracts subject to delayed (10 min after tissue disruption) pH control (initial extract pH of 5.2–5.4) displayed the greatest extent of discoloration, in descending order, at pH 4.0 > 5.0, 6.0 > 3.0. In these samples, initial thiosulfinate levels were similar and the greatest residual thiosulfinate levels over the 21-day incubation period were observed at pH 3.0 and 4.0 (about 50% of maximum levels), with the lowest residual levels observed for extracts adjusted to pH 6.0 (about 20% of maximum levels). Tissue extracts supplemented with 9- to 18-fold excess MCSO were subject to modest increases in both extent of discoloration and thiosulfinate levels.     

Estimation of Aspergillus flavus Growth under the Influence of Different Formulation Factors by Means of Kinetic,Probabilistic, and Survival Models

A Box-Behnken design was conducted to determine the effect of casein concentration (0, 5, or 10%), corn oil (0, 3, or 6%), a_w (0.900, 0.945, or 0.990), pH (3.5, 5.0, or 6.5), concentration of cinnamon essential oil (CEO: 0, 200, or 400 ppm), and incubation temperature (15, 25, or 35 °C) on the growth of A. flavus during 50 days of incubation. Potato dextrose agars were adjusted to the different levels of tested factors and poured into Petri dishes, once solidified were inoculated with mold spores and incubated at studied temperatures. Mold response was modeled using Gompertz and quadratic polynomial equations. The obtained polynomial regression model (allowed the significant (p<0.05) for linear, quadratic, and interaction effects for the Gompertz equation coefficients’ parameters to be identified) adequately described (R²>0.97) mold growth. Additionally, in order to describe growth/not-growth boundary, collected data after 50 days of incubation were classified according to the observed response as 1 (growth) or 0 (not growth), then a binary logistic regression was used to model growth interface. Mold growth probability strongly depend on casein, oil, temperature, and a_w, as well as variations of pH and CEO concentration, being lower for those systems with higher content of CEO (>180 ppm). Furthermore, survival analysis using failure time was utilized to estimate the time at which mold growth began. The time to fail was directly related to the temperature and CEO concentration; for systems formulated with more than 200 ppm of CEO, time to fail was >30 days for low protein and fat contents. The three tested approaches to describe A. flavus response, adequately predicted growth rate and lag time, or growth probability, or the time in which growth will occur. The use and selection of any of these approaches will depend on the intended application.     

Prevention of Aspergillus ochraceus growth on and Ochratoxin a contamination of sausages using ozonated air

Mycotoxigenic moulds can grow on the surface of sausages and reduce the safety of these sausages for consumption. The aim of this study was to prevent the growth of Aspergillus ochraceus and the presence of Ochratoxin A (OTA) on the surface of Milano-type sausages using ozonated air. Spores of A. ochraceus were used to inoculate the casings of the sausages after casing. A portion of the lot (35 samples) was ripened in typical rooms, and another portion (35 samples) was dried and ripened in a separate room that was treated with gaseous ozone. The gas was delivered at night (8 h/day) at a concentration of ∼1 ppm. The temperature and relative humidity during the drying and ripening were the same for both rooms. Our results demonstrate that the gaseous ozone treatment prevented the growth of A. ochraceus and, consequently, the presence of OTA. In contrast, A. ochraceus grew and produced OTA on the untreated sausages. Moreover, the use of ozone did not influence the ripening, physico-chemical parameters, peroxide value or sensorial characteristics of the sausages.