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.     

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.     

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.     

The impact of fullerenol nanoparticles on the growth of toxigenic Aspergillus flavus and aflatoxins production in vitro and in corn flour

Antifungal and antimycotoxigenic activity of fullerenol nanoparticles (FNPs) were investigated on Aspergillus flavus growth isolated from a real food sample and aflatoxins (AFs) (AFB₁ and AFB₂) production. The final FNPs concentrations in in vitro and in commercial corn flour after the stationary incubation period of 7 and 14 days were in the range 0.16–80 µg/mL and 0.16–80 µg/g, respectively. Nanocharacterization of FNPs revealed an average size of 5–20 nm and a zeta potential of −35 mV. The highest degree of A. flavus mycelium growth inhibition (28%) after 7 days was observed for applied FNP concentration of 8.0 µg/mL, while after 14 days FNP concentration of 0.32 µg/mL led to the maximal inhibition of A. flavus mycelium growth (36%). Spearman's correlations analysis revealed a strong positive correlation between AFB₁ and AFB₂ concentrations in YES broth after 7 (R = 0.994, p < 0.05) and 14 days (R = 0.976), as well as between AFs concentrations and A. flavus mycelium mass after 7 (R = 0.786 for AFB₁ and R = 0.766 for AFB₂) and 14 days (R = 0.810 for AFB₁ and R = 0.833 for AFB₂). Paired samples t-test showed the existence of a statistically significant difference (p < 0.05) between the produced AFs concentrations after the incubation of 7 and 14 days. Regarding the artificially inoculated corn flour the lower applied FNP concentrations (0.16–0.8 µg/g) achieved a reduction of AFB₁ up to 42% and 60% after 7 and 14 days, respectively.     

Assessment of azole fungicides as a tool to control growth of Aspergillus flavus and aflatoxin B1 and B2 production in maize

ABSTRACT

Aspergillus flavus is a highly aflatoxin (AF)-producing species infecting maize and other crops. It is dominant in tropical regions, but it is also considered an emerging problem associated with climate change in Europe. The aim of this study was to assess the efficacy of azole fungicides (prochloraz, tebuconazole and a 2:1 (w/w) mixture of prochloraz plus tebuconazole) to control the growth of A. flavus and AF production in yeast-extract–sucrose (YES) agar and in maize kernels under different water activities (a_w) and temperatures. Aflatoxins B₁ and B₂ were determined by LC with fluorescence detection and post-column derivatisation of AFB₁. In YES medium and maize grains inoculated with conidia of A. flavus, the growth rate (GR) of the fungus and AFB₁ and AFB₂ production were significantly influenced by temperature and treatment. In YES medium and maize kernels, optimal temperatures for GR and AF production were 37 and 25°C, respectively. In maize kernels, spore germination was not detected at the combination 37ºC/0.95 a_w; however, under these conditions germination was found in YES medium. All fungicides were more effective at 0.99 than 0.95 a_w, and at 37 than 25ºC. Fungicides effectiveness was prochloraz > prochloraz plus tebuconazole (2:1) > tebuconazole. AFs were not detected in cultures containing the highest fungicide doses, and only very low AF levels were found in cultures containing 0.1 mg l^–1 prochloraz or 5.0 mg l^–1 tebuconazole. Azoles proved to be highly efficient in reducing A. flavus growth and AF production, although stimulation of AF production was found under particular conditions and low-dosage treatments. Maize kernels were a more favourable substrate for AF biosynthesis than YES medium. This paper is the first comparative study on the effects of different azole formulations against A. flavus and AF production in a semi-synthetic medium and in maize grain under different environmental conditions.     

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.     

Distribution of fungi and aflatoxins in a stored peanut variety

The objective of the present study was to evaluate the mycoflora and occurrence of aflatoxins in stored peanut samples (hulls and kernels) from Tupã, State of São Paulo, Brazil. The samples were analyzed monthly over a period of one year. The results showed a predominance of Fusarium spp. (67.7% in hulls and 25.8% in kernels) and Aspergillus spp. (10.3% in hulls and 21.8% in kernels), and the presence of five other genera. The growth of Aspergillus flavus was mainly influenced by temperature and relative humidity. Analysis of hulls showed that 6.7% of the samples were contaminated with AFB₁ (mean levels = 15–23.9 μg/kg) and AFB₂ (mean levels = 3.3–5.6 μg/kg); in kernels, 33.3% of the samples were contaminated with AFB₁ (mean levels = 7.0–116 μg/kg) and 28.3% were contaminated with AFB₂ (mean levels = 3.3–45.5 μg/kg). Analysis of the toxigenic potential revealed that 93.8% of the A. flavus strains isolated were producers of AFB₁ and AFB₂.     

The control of Aspergillus flavus with Cinnamomum jensenianum Hand.-Mazz essential oil and its potential use as a food preservative

The essential oil extracted from the bark of Cinnamomum jensenianum Hand.-Mazz was tested for antifungal activity against Aspergillus flavus. Fifty-five components accounting for 96.66% of the total oil composition were identified by GC–MS. The major components were eucalyptol (17.26%) and α-terpineol (12.52%). Mycelial growth and spore germination was inhibited by the oil in a dose-dependent manner. The oil also exhibited a noticeable inhibition on the dry mycelium weight and the synthesis of aflatoxin B₁ (AFB₁) by A. flavus, completely restraining AFB₁ production at 6 μl/ml. The possible mode of action of the oil against A. flavus is discussed based on changes in the mycelial ultrastructure. To confirm the target of the oil in the plasma membrane, studies on the inhibition of ergosterol synthesis were performed. Results show that the oil caused a considerable reduction in the ergosterol quantity. Thus, the essential oil from C. jensenianum can be used as a potential source for food preservative.     

Growth kinetics of Listeria monocytogenes and spoilage microorganisms in fresh-cut cantaloupe

The main objective of this study was to investigate the growth kinetics of Listeria monocytogenes and background microorganisms in fresh-cut cantaloupe. Fresh-cut cantaloupe samples, inoculated with three main serotypes (1/2a, 1/2b, and 4b) of L. monocytogenes, were incubated at different temperatures, ranging from 4 to 43 °C, to develop kinetic growth models. During storage studies, the population of both background microorganisms and L. monocytogenes began to increase almost immediately, with little or no lag phase for most growth curves. All growth curves, except for two growth curves of L. monocytogenes 1/2a at 4 °C, developed to full curves (containing exponential and stationary phases), and can be described by a 3-parameter logistic model. There was no significant difference (P = 0.28) in the growth behaviors and the specific growth rates of three different serotypes of L. monocytogenes inoculated to fresh-cut cantaloupe. The effect of temperature on the growth of L. monocytogenes and spoilage microorganisms was evaluated using three secondary models. For L. monocytogenes, the minimum and maximum growth temperatures were estimated by both the Ratkowsky square-root and Cardinal parameter models, and the optimum temperature and the optimum specific growth rate by the Cardinal parameter model. An Arrhenius-type model provided more accurate estimation of the specific growth rate of L. monocytogenes at temperatures <4 °C. The kinetic models developed in this study can be used by regulatory agencies and food processors for conducting risk assessment of L. monocytogenes in fresh-cut cantaloupe, and for estimating the shelf-life of fresh-cut products.     

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.     

Modified atmosphere influences aflatoxin B1 contamination and quality of peanut (Arachis hypogaea L.) kernels cv. Khon Kaen 84-8

Storage of ‘Khon Kaen 84-8’ peanut kernels in laminated bags (Low density polyethylene (LDPE)/Nylon) (20×30 cm; 120 μm thickness) under different packaging atmospheres had effect on quality, fungal occurrence and aflatoxin B₁ (AFB₁) contamination. During fifteen week storage, peanuts (6.4% moisture content) were kept under different packaging atmospheres with some inoculated (Inoc.) and without (Non-Inoc.) aflatoxin producing fungus Aspergillus flavus. The treatments include; 100% CO₂+Inoc., 100% N₂+Inoc., vacuum + Inoc., Air + Inoc., and Air + Non-Inoc. packed with only ambient air. The storage room conditions were a temperature of 29±2 °C and 70 ± 5% r. h. Mycobiota occurrence, AFB₁ level, moisture content, lipase activity, free fatty acids and rancidity (Thiobarbituric acid; TBA value) were investigated. A total of five major fungi were identified with four storage fungi and one field fungus; Rhizopus stolonifer, Aspergillus niger, A. flavus, Penicillium spp., and Fusarium spp., respectively. Packaging in 100% CO₂+Inoc. significantly repressed the fungal occurrence especially A. niger while all other packaging atmospheres suppressed proliferation of both R. stolonifer and Penicillium spp. The peanuts kept in vacuum + Inoc. package revealed AFB₁ level below detection limit (0.4 μg/kg). Peanut kernels in 100% CO₂+Inoc. packaging atmosphere maintained an acceptable color indicated by L* and a* values, as well as the color change (ΔE) compared to other treatments. Different packaging atmospheres showed variable results in relation to the free fatty acids level with packaging under 100% N₂ revealing the lowest. Subsequently, 100% N₂ packaging atmosphere relatively inhibited rancidity occurrence in peanut kernels during storage. The results of this study portray that 100% CO₂, 100% N₂ and vacuum packaging atmosphere could have a potential to suppress occurrence of mycobiota, maintain peanut kernel quality in relation to color and lipid oxidation, and as well curtail AFB₁ contamination respectively, for fifteen weeks or more given the quality of kernels at the end of storage.     

Effect of pre-incubation conditions on growth and survival of Staphylococcus aureus in sliced cooked chicken breast

In this work, the effect of pre-incubation conditions (temperature: 10, 15, 37 °C; pH 5.5, 6.5 and water activity, a_w: 0.997, 0.960) was evaluated on the subsequent growth, survival and enterotoxin production (SE) of Staphylococcus aureus in cooked chicken breast incubated at 10 and 20 °C. Results showed the ability of S. aureus to survive at 10 °C when pre-incubated at low a_w (0.960) what could constitute a food risk if osmotic stressed cells of S. aureus which form biofilms survive on dried surfaces, and they are transferred to cooked meat products by cross-contamination. Regarding growth at 20 °C, cells pre-incubated at pH 5.5 and a_w 0.960 had a longer lag phase and a slower maximum growth rate. On the contrary, it was highlighted that pre-incubation at optimal conditions (37 °C/pH 6.5/a_w 0.997) produced a better adaptation and a faster growth in meat products what would lead to a higher SE production. These findings can support the adoption of management strategies and preventive measures in food industries leading to avoid growth and SE production in meat products.     

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.     

The application of novel rotary plasma jets to inhibit the aflatoxin-producing Aspergillus flavus and the spoilage fungus,Aspergillus niger on peanuts

The major safety risk of peanuts is contamination with aflatoxin. Cold atmospheric plasma (CAP) has been demonstrated to inactivate fungi effectively. In this study, a novel CAP device with a rotary jet system was used to inactivate the existing A. flavus and A. niger on peanuts. The initial inoculation levels were 6.39 and 5.83 log CFU/g of A. flavus and A. niger, respectively. After treatments at 180 W for 7.5 min and 200 W for 5 min, A. flavus was not detected. For A. niger, the treatments at 180 W for 10 min and 200 W for 5 min resulted in undetected population. Observation under scanning electron microscope revealed the fungal spores were evidently damaged. The growth of A. flavus and aflatoxin concentrations were significantly lower (p < 0.05) on the group treated with 200 W for 5 min than other treatment groups stored for 29 d. Oil quality indexes of the CAP-treated peanuts were maintained in the range of excellent grades. This study demonstrated CAP effectively inhibited fungal growth and toxin production without adversely affected oil quality.     

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.     

Microbial inactivation after high-pressure processing at 600 MPa in commercial meat products over its shelf life

The behaviour of spoilage and pathogenic microorganisms was evaluated after high-pressure treatment (600 MPa 6 min, 31 °C) and during chilled storage at 4 °C for up to 120 days of commercial meat products. The objective was to determine if this pressure treatment is a valid process to reduce the safety risks associated with Salmonella and Listeria monocytogenes, and if it effectively avoids or delays the growth of spoilage microorganisms during the chilled storage time evaluated. The meat products covered by this study were cooked meat products (sliced cooked ham, pH 6.25, a_w 0.978), dry cured meat products (sliced dry cured ham, pH 5.81, a_w 0.890), and raw marinated meats (sliced marinated beef loin, pH 5.88, a_w 0.985). HPP at 600 MPa for 6 min was an efficient method for avoiding the growth of yeasts and Enterobacteriaceae with a potential to produce off-flavours and for delaying the growth of lactic acid bacteria as spoilage microorganisms. HPP reduced the safety risks associated with Salmonella and L. monocytogenes in sliced marinated beef loin.     

Inhibitory effect of sour orange (Citrus aurantium) juice on Salmonella Typhimurium and Listeria monocytogenes

The sour orange (Citrus aurantium) juice is commonly used as flavoring and acidifying agent for vegetable salads and appetizers in Turkey. It was aimed to determine the survival and growth pattern of Salmonella Typhimurium and Listeria monocytogenes in sour orange juice. Different concentrations of neutralized and un-neutralized juice samples were inoculated with each of the test microorganisms (∼6 log CFU/mL) separately and then incubated at 4 °C and 37 °C for seven days. It was detected both of the test microorganisms could survive and even grow in neutralized juice samples at 37 °C for two days. However, none of them could survive at the end of seventh day of incubation at 37 °C. Low incubation temperature (+4 °C) increased the survival of the tested microorganisms. Also, it was detected that L. monocytogenes were less resistant to the variable conditions than S. Typhimurium. It was concluded that the antimicrobial effect of sour orange juice mainly depends on the low pH value of the product. However, incubation time and temperature are also effective on the survival of the tested pathogens.     

Effect of CO2 modified atmosphere packaging on aflatoxin production in maize infested with Sitophilus zeamais

The weevil Sitophilus zeamais (Motschulsky), the maize weevil, is a pest of stored maize that can cause feeding damage and lead to the proliferation of toxigenic fungi. The application of modified atmospheres with a high concentration of CO₂ is an alternative method for the control of S. zeamais and the inhibition of fungal growth. The objectives of the study were to determine the effect of S. zeamais infestation, grain damage and grain moisture content on aflatoxin production by Aspergillus flavus on maize, and the impact of high CO₂ modified atmosphere packaging on pest infestation and aflatoxin production. Mycotoxin production was only recorded when maize was infested with S. zeamais and had A. flavus inoculum. However, production of mycotoxins was not recorded when the maize was mechanically damaged and stored at 18% moisture content, indicating that the biological activity of the insect was determinant in the production of mycotoxins. The high CO₂ modified atmosphere packaging tested (90% CO₂, 5% O₂ and 5% N₂) prevented mycotoxin production.     

Increase in aflatoxins due to Aspergillus section Flavi multiplication during the aerobic deterioration of corn silage treated with different bacteria inocula

The growth of Aspergillus flavus and the production of aflatoxins (AF) during the aerobic deterioration of corn silage represent a problem for animal and human health. This experiment was conducted to evaluate whether the presence of A. flavus and AF production originate from the field or additional AF are produced during the fermentation phase or during aerobic deterioration of corn silage. The trial was carried out in northern Italy on corn at a dry matter (DM) level of 34%. The fresh herbage was either not treated (C) or treated with a Lactobacillus buchneri (LB) NCIMB 40788 [(at 3 × 10⁵ cfu/g of fresh matter (FM)], Lactobacillus hilgardii (LH) CNCM I-4785 (at 3 × 10⁵ cfu/g of FM), or their combination (LB+LH; at 1.5 × 10⁵ cfu/g of FM of each strain) ensiled in 20-L silos and opened after 250 d of ensiling. After silo opening, the aerobic stability was evaluated and samples were taken after 7 and 14 d of air exposure. The pre-ensiled material, the silages at silo opening, and the aerobically exposed silages were analyzed for DM content, fermentative profiles, microbial count, nutritive characteristics, DM losses, and AFB₁, AFB₂, AFG₁, and AFG₂ contents. Furthermore, a subsample of colonies with macromorphological features of Aspergillus section Flavi was selected for AF gene pattern characterization and in vitro AF production. The presence of A. flavus was below the detection limit (<1.00 log₁₀ cfu/g) in the fresh forage before ensiling, whereas it was found in 1 out of 16 silage samples at silo opening at a level of 1.24 log₁₀ cfu/g. The AF were found in both the fresh forage and at opening in all the samples, with a predominance of AFB₂ (mean value of 1.71 μg/kg of DM). The inoculation of lactic acid bacteria determined a reduction in the lactic-to-acetic ratio compared with the control. A larger amount of acetic acid resulted in a lower yeast count and higher aerobic stability in the treated silages than in the control ones. At the beginning of aerobic deterioration, the yeasts increased to over 5 log₁₀ cfu/g, whereas the molds were close to the value observed at silo opening. When the inhibiting conditions were depleted (pH and temperature higher than 5 and 35°C, respectively), both the total molds and A. flavus reached higher values than 8.00 and 4.00 log₁₀ cfu/g, respectively, thus determining the ex novo production of AFB₁ during aerobic deterioration, regardless of treatments. The analysis of gene pattern showed that 64% of the selected colonies of A. flavus showed the presence of all 4 AF gene patterns, and 43% of the selected colonies were able to produce AF in vitro. During air exposure, after 1,000°C·h have been cumulated, starch content decreased (below 10% DM) and concentration of neutral detergent fiber, acid detergent fiber, hemicelluloses, crude protein, and ash increased. The inoculation with LB and LB+LH increased the aerobic stability of the silages and delayed the onset of aerobic microbial degradation, which in turn indirectly reduced the risk of A. flavus outgrowth and AFB₁ production after silage opening.