Effect of aw and CO2 level on Aspergillus flavus growth and aflatoxin production in high moisture maize post-harvest

The potential for using modified atmospheres of 25-75% CO2 (balanced with N2) and water activity (aw, 0.95, 0.92) to control Aspergillus flavus development and aflatoxin B1 production has been evaluated (a) on synthetic medium and (b) on maize grain during storage for up to 21 days at 25 degrees C. On agar medium up to 75% CO2 at both 0.95 and 0.92 aw significant inhibition of growth was obtained (P<0.05). In stored grain inoculated with spores of A. flavus there was significantly higher populations of the species at 0.95 aw than 0.92 aw. Up to 75% CO2 resulted in an inhibition of the populations of A. flavus isolated from the grain. Contrasting aflatoxin B1 production was obtained on agar and in stored maize grain. On agar, greatest amounts were produced at 0.92 aw, while more was produced at 0.95 aw on maize grain. Overall, the efficacy of controlled atmospheres x aw showed that treatment with 25% CO2 could be sufficient to efficiently reduce A. flavus development but at least 50% CO2 was required to obtain a significant reduction of aflatoxin synthesis.     

Occurrence of mycotoxins (ochratoxin A, deoxynivalenol) and toxigenic fungi in Moroccan wheat grains: impact of ecological factors on the growth and ochratoxin A production

The aim of the present work was to evaluate the contamination of some samples, taken from Moroccan wheat grains, by ochratoxin A (OTA), deoxynivalenol (DON) and the associated toxigenic fungi. Moreover, we focused on the influence of environmental factors on both the growth and OTA production by three strains of Aspergillus. The results showed that only few samples were contaminated by the two mycotoxins (2 samples for OTA and 7 for DON). The main isolated fungi belong to the Aspergillus, Penicillium and Fusarium genus; 74 Aspergillus and 28 Penicillium isolates were tested for their ability to produce OTA. Only 2 A. alliaceus and 14 A. niger were able to synthesize OTA. However, none of Penicillium isolates can produce this toxin under the conditions mentioned. In respect of the effects of the temperature and water activity (aw), the optimal conditions for the growth and OTA production were different. While the optimal conditions of growth for A. alliaceus and A. terreus are 30 degrees C and 0.98 aw, A. niger preferred 0.93-0.95 aw at 25 degrees C, whereas the optimal production of OTA was observed at 30 degrees C for both A. alliaceus and A. niger at 0.93 and 0.99 aw, respectively.     

Natural maize phenolic acids for control of aflatoxigenic fungi on maize

ABSTRACT:  Natural phytochemicals may be an alternative to synthetic chemicals for controlling fungal growth and mycotoxin production in stored maize. A key to progress in this field is to select the best natural maize phytochemicals to be applied in a storage maize ecosystem. This research was undertaken to evaluate the effects of the natural phytochemicals trans-cinnamic acid (CA) and ferulic acid (FA) alone at concentrations of 20 to 30 mM and in 5 combinations on Aspergillus flavus Link and A. parasiticus Speare populations and aflatoxin B₁ production. Studies on Aspergillus population and aflatoxin B₁ production were carried out in maize grain in relation to a water activity a_w of 0.99, 0.97, 0.95, and 0.93. CA and FA at concentrations of 25 to 30 mM, respectively, and CA-FA mixture T9 (25 + 30 mM) were the treatments most effective at inhibiting A. flavus and A. parasiticus population at all a_w assayed after 11 d of incubation. At all a_w values, the mixture CA-FA T9 (25 + 30 mM) completely inhibited (100%) aflatoxin B₁ production by both strains at a_w= 0.99, 0.97, 0.95, and 0.93. Decreased aflatoxin B₁ levels in comparison with the control were observed with mixtures CA-FA T6 (10 + 25 mM), T7 (20 + 20 mM), and T8 (20 + 30 mM) of both strains in the majority of a_w assayed. The data show that CA and FA could be considered as effective fungitoxicants for A. flavus and A. parasiticus in maize in the a_w range 0.99 to 0.93. The information obtained shows promise for controlling aflatoxigenic fungi in stored maize.     

Studies on Aspergillus section Flavi isolated from maize in northern Italy

In 2003, for the first time in Italy, significant problems arose with colonization and contamination of maize destined for animal feed with Aspergillus section Flavi and aflatoxins (AFs). This resulted in milk and derived products being contaminated with AFM(1) at levels above the legislative limit. There was little knowledge and experience of this problem in Italy. The objectives of this research were thus to study the populations of Aspergillus section Flavi in six northern Italian regions and obtain information on the relative role of the key species, ability to produce sclerotia, production of the main toxic secondary metabolites, aflatoxins and cyclopiazonic acid, and tolerance of key environmental parameters. A total of 70 strains were isolated and they included the toxigenic species A. flavus and A. parasiticus. A. flavus was dominant in the populations studied, representing 93% of the strains. Seventy percent of strains of Aspergillus section Flavi produced AFs, with 50% of strains also producing cyclopiazonic acid. Sixty-two percent of A. flavus strains and 80% of A. parasiticus were able to produce sclerotia at 30 degrees C. Using 5/2 agar, only 1 strain developed S sclerotia and 19 L sclerotia. With regard to ecological studies, growth of Aspergillus section Flavi was optimal at between 25 and 30 degrees C, while AFB(1) production was optimal at 25 degrees C. Regarding water availability (water activity, a(w)), 0.99 a(w) was optimal for both growth and AFs production, while the only aflatoxin produced in the driest condition tested (0.83 a(w)) was AFB(1). This information will be very useful in identifying regions at risk in northern Italy by linking climatic regional information to levels of fungal contamination present and potential for aflatoxin production in maize destined for animal feed. This would be beneficial as part of a prevention strategy for minimising AFs in this product.     

Inhibition of aflatoxin-producing fungi by Welsh onion extracts.

Welsh onion ethanol extracts were tested for their inhibitory activity against the growth and aflatoxin production of Aspergillus flavus and A. parasiticus. The survival of spores of A. flavus and A. parasiticus depended on both the extract concentration and the exposure time of the spores to the Welsh onion extracts. The mycelial growth of two tested fungi cultured on yeast extract-sucrose broth was completely inhibited in the presence of the Welsh onion ethanol extract at a concentration of 10 mg/ml during 30 days of incubation at 25 degrees C. The extracts added to the cultures also inhibited aflatoxin production at a concentration of 10 mg/ml or permitted only a small amount of aflatoxin production with extract concentration of 5 mg/ml after 2 weeks of incubation. Welsh onion ethanol extracts showed more pronounced inhibitory effects against the two tested aflatoxin-producing fungi than did the same added levels of the preservatives sorbate and propionate at pH values near 6.5.     

EFFECT OF GAMMA IRRADIATION ON AFLATOXIN B1 PRODUCTION BY ASPERGILLUS FLAVUS IN GROUND BEEF STORED AT 5C

The effect of γ-irradiation for controlling the production of aflatoxin B₁ by Aspergillus flavus in ground beef stored at 5C for 2 weeks was investigated. Aspergillus, Penicillium, Cladosporium, Mucor, Scopulariopsis, Candida and Rhodotorula were the most common fungal genera contaminating ground beef. A. flavus and A. niger were the most common Aspergillus spp. Aspergillus flavus isolates were able to produce aflatoxin B₁ in ground beef. Only 3 (20%) samples of ground beef were contaminated with aflatoxin B₁ (25–45 μg/Kg). Gamma irradiation dose levels resulted in an immediate reduction in the total numbers of A. flavus. No growth or aflatoxin B₁ production occurred at 1.50 kGy during storage.     

Influence of gamma-irradiation and maize lipids on the production of aflatoxin B1 by Aspergillus flavus

The effect of gamma-irradiation and maize lipids on aflatoxin B1 production by Aspergillus flavus artificially inoculated into sterilized maize at reduced water activity (aw 0.84) was investigated. By increasing the irradiation doses the total viable population of A. flavus decreased and the fungus was completely inhibited at 3.0 kGy. The amounts of aflatoxin B1 were enhanced at irradiation dose levels 1.0 and 1.5 kGy in both full-fat maize (FM) and defatted maize (DM) media and no aflatoxin B1 production at 3.0 kGy gamma-irradiation over 45 days of storage was observed. The level in free lipids of FM decreased gradually, whereas free fatty acid values and fungal lipase activity increased markedly by increasing the storage periods. The free fatty acid values decreased by increasing the irradiation dose levels and there was a significant enhancement of fungal lipase activity at doses of 1.0 and 1.50 kGy. The ability of A. flavus to grow at aw 0.84 and produce aflatoxin B1 is related to the lipid composition of maize. The enhancement of aflatoxin B1 at low doses was correlated to the enhancement of fungal lipase activity.     

Influence of water activity and temperature on the accumulation of zearalenone in corn

The influence of water activity (aw) and temperature on the zearalenone biosynthesis in corn has been examined. Viable corn kernels were conditioned at different values of water activity (0.90, 0.95 and 0.97), inoculated with Fusarium graminearum and incubated at different temperatures. Zearalenone was determined at selected times. For the strain used, a constant temperature of 25 degrees C resulted more favorable than 15 degrees C, 20 degrees C and the combination of two weeks at 25 degrees C followed by incubation at 15 degrees C shown by other authors to increase the zearalenone accumulation. Both F. graminearum growth and zearalenone production at 25 degrees C were inhibited at water activity 0.90. At short incubation times, toxin accumulation was greater at water activity 0.97 than at water activity 0.95 (25 degrees C). This relation was inverted at longer periods of incubation. A combined effect of water activity and temperature on the zearalenone accumulation was observed.     

Study of the effect of water activity and temperature on ochratoxin A production by Aspergillus carbonarius

The effect of water activity (aw) (0.78-0.99) and temperature (15 and 30 degrees C) on growth and production of ochratoxin A (OTA) of six Aspergillus carbonarius strains was studied in two culture media: Czapek yeast autolysate (CYA) agar and yeast extract sucrose (YES) agar, during a period of 30 days. The strains were selected to include different sources and different reported abilities to produce OTA and were characterized by RAPD and ITS-5.8S rDNA sequencing. CYA showed to be better culture medium than YES for OTA production in the isolates tested. OTA concentration was higher at 15 degrees C than at 30 degrees C. At 30 degrees C, ranges for OTA production were more restrictive than those for growth. OTA was produced from 0.86, 0.90 or 0.94 aw depending on the strain. At 15 degrees C, growth and OTA production were detected only in the 0.94-0.99 aw range. The molecular study performed showed that five of the strains were conspecific and no correlation was found between molecular data and the OTA production level or origin. The remaining strain had never been able to produce OTA and will probably represent a new species in the Aspergillus section Nigri. Our results show that A. carbonarius is able to grow and produce OTA in a wide range of water activities at both high and low temperatures.     

Impact of environment and interspecific interactions between spoilage fungi and Aspergillus ochraceus on growth and ochratoxin production in maize grain

Using layers of irradiated but still fertile maize grain, the effects of water activity (0.995, 0.95 a(w)) and temperature (18, 30 degrees C) on interspecific interactions between Aspergillus ochraceus and five other spoilage fungi were examined. Asp. ochraceus was not competitive against Asp. flavus, Asp. niger, or Alternaria alternata at 18 degrees C when water was freely available (0.995 a(w)), while at 0.95 a(w) it was dominant against Asp. candidus, Asp. flavus and Alt. alternata. At 30 degrees C and 0.995 a(w), Asp. ochraceus was dominated by other fungi, except Alt. alternata, and was mutually antagonistic to Asp. candidus and Eurotium amstelodami. However, at 30 degreees C and 0.95 a(w), it was competitive against Asp. candidus and Alt. alternata, but not against the other species examined. The overall Index of Dominance showed that Asp. ochraceus was not competitive under the conditions examined here. At 18 degrees C ochratoxin production by Asp. ochraceus was inhibited significantly by Asp. candidus (0.995 and 0.95 a(w)) and Asp. niger (0.995 a(w)). When grown on maize grain at 30 degrees C, ochratoxin production by Asp. ochraceus was significantly inhibited by other spoilage fungi when both were grown on maize grain, especially by Asp. niger and E. amstelodami (0.995 a(w)) and Asp. flavus at 0.95 a(w). These results suggest that, to a large extent, A. ochraceus is not as competitive as some other spoilage fungi in primary resource capture on maize grain at a(w) of 0.95 or above, although it may modify resource quality and influence secondary colonisation by other species under the conditions tested.     

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.     

Influence of temperature,water activity and pH on growth of some xerophilic fungi

The combined effects of water activity (aw), pH and temperature on the germination and growth of seven xerophilic fungi important in the spoilage of baked goods and confectionery were examined. Eurotium rubrum, E. repens, Wallemia sebi, Aspergillus penicillioides, Penicillium roqueforti, Chrysosporium xerophilum and Xeromyces bisporus were grown at 25, 30 and 37 degrees C on media with pH values of 4.5, 5.5, 6.5 and 7.5 and a range of water activities (aw) from 0.92 to 0.70. The aw of the media was controlled with a mixture of equal parts of glucose and fructose. Temperature affected the minimum aw for germination for most species. For example, P. roqueforti germinated at 0.82 aw at 25 degrees C, 0.86 aw at 30 degrees C and was unable to germinate at 37 degrees C. E. repens germinated at 0.70 aw at 30 degrees C, but at 25 and 37 degrees C, its minimum aw for germination was 0.74. C. xerophilum and X. bisporus germinated at 0.70 aw at all three temperatures. The optimum growth occurred at 25 degrees C for P. roqueforti and W. sebi, at 30 degrees C for Eurotium species, A. penicillioides and X. bisporus and at 37 degrees C for C. xerophilum. These fungi all grew faster under acidic than neutral pH conditions. The data presented here provide a matrix that will be used in the development of a mathematical model for the prediction of the shelf life of baked goods and confectionery.     

Modified atmosphere packaging for prevention of mold spoilage of bakery products with different pH and water activity levels

A sponge cake analog was used to study the influence of pH, water activity (aw), and carbon dioxide (CO2) levels on the growth of seven fungal species commonly causing bakery product spoilage (Eurotium amstelodami, Eurotium herbariorum, Eurotium repens, Eurotium rubrum, Aspergillus niger, Aspergillus flavus, and Penicillium corylophilum). A full factorial design was used. Water activity, CO2, and their interaction were the main factors significantly affecting fungal growth. Water activity at levels of 0.80 to 0.90 had a significant influence on fungal growth and determined the concentration of CO2 needed to prevent cake analog spoilage. At an aw level of 0.85, lag phases increased twofold when the level of CO2 in the headspace increased from 0 to 70%. In general, no fungal growth was observed for up to 28 days of incubation at 25 degrees C when samples were packaged with 100% CO2, regardless of the aw level. Partial least squares projection to latent structures regression was used to build a polynomial model to predict sponge cake shelf life on the basis of the lag phases of all seven species tested. The model developed explained quite well (R2 = 79%) the growth of almost all species, which responded similarly to changes in tested factors. The results of this study emphasize the importance of combining several hurdles, such as modified atmosphere packaging, aw, and pH, that have synergistic or additive effects on the inhibition of mold growth.     

Water activity influence on aflatoxin accumulation in corn

The influence of water activity on the production of B1, B2, G1 and G2 aflatoxins in corn has been examined. Viable corn kernels were conditioned at three water activity levels (0.87, 0.90 and 0.97) and inoculated with Aspergillus parasiticus at 30 degrees C. Aflatoxin accumulation was determined at selected times by thin-layer chromatography. For the strain used total aflatoxin accumulation was greater at water activity 0.90 than at 0.87 and 0.97 for the incubation times studied.     

Influence of aw value and storage temperature on the multiplication and enterotoxin formation of staphylococci in dry-cured raw hams.

Growth and enterotoxin A production in dry-cured raw ham, with different aw and pH values, was investigated for Staphylococcus aureus strain FRJ-100 over a temperature range of 20-35 degrees C when stored for up to 7 days. Enterotoxin production took place at all temperatures tested in the meat of ham that was cured, but not dried (aw 0.95). In dry-cured raw ham with an aw-value of about 0.92, and stored at 20 degrees C, no enterotoxin production was detectable within the 7-day storage period, whereas enterotoxin was produced at the higher temperatures. In ham with an aw of about 0.89, enterotoxin was produced only when stored for 7 days at 35 degrees C. Critical points for S. aureus multiplication and enterotoxin-formation during production and storage of dry-cured raw ham are discussed.     

Effect of different postharvest drying temperatures on Aspergillus flavus survival and aflatoxin content in five maize hybrids

After harvest, maize is dried artificially to halt fungal growth and mycotoxin production while in postharvest storage. The process often limits harvest capacity and has been a frequent cause of seed injury. Higher drying temperatures could lead to shorter drying periods and faster turnover; however, there is often a deterioration of the physical grain quality, including increased breakage susceptibility and loss of viability. The goals of this study were to determine the effect of different postharvest drying temperatures on Aspergillus filavus and Fusarium verticillioides survival and aflatoxin content in maize and to determine the viability of the seed. Five corn hybrids varying in resistance to A. flavus were side needle-inoculated with A. flavus, harvested at physiological maturity, and dried at temperatures ranging from 40 to 70 degrees C. Kernels were evaluated for aflatoxin, stress cracks, germination, and kernel infection by A. flavus and a natural infestation of F. verticillioides. Drying temperature had no effects on aflatoxin concentration given the heat stability of the toxin. With increased temperatures from 40 to 70 degrees C, germination decreased significantly, from 96 to 27%, and stress cracks increased significantly (1.4 up to 18.7). At temperatures above 60 degrees C, F. verticillioides kernel infection was significantly reduced to less than 18%. At 70 degrees C, there was a significant reduction in A. flavus kernel infection, from 11 to 3%. This information is useful in determining a range of temperatures that can be used for drying seed when fungal infection, stress cracks, and seed viability are of interest.     

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.     

INFLUENCE OF MOISTURE CONTENT AND STORAGE TEMPERATURE ON THE PRODUCTION OF AFLATOXIN BY ASPERGILLUS FLAVUS EA-81 IN MAIZE AFTER EXPOSURE TO GAMMA RADIATION

The effect of gamma radiation on aflatoxin production by Aspergillus flavus EA-81 in maize with different initial moisture levels was determined over a 15-day period. The viability of A. flavus on maize decreased over time with increasing moisture contents and storage at 8C. After 45 days at 28C, levels of viable conidiospores of A. flavus increased from 4.5 × 10⁷ to about 3.0 × 10⁸ per gram of maize. Levels of aflatoxin B₁ produced by A. flavus were 10 μg kg^-1 in the maize stored at 8C after 45 days. Production of aflatoxin was highest at 40% moisture and 28C. Irradiation of 1.0 or 2.0 kGy greatly reduced the level of mold growth relative to unirradiated controls. A dose of 4.0 kGy eliminated all viable fungi. Aflatoxin B₁ production decreased with increased levels of irradiation and was negligible at 4.0 kGy. When maize was inoculated after irradiation and stored, the spore counts and aflatoxin levels were higher than in unirradiated and inoculated controls after 30 days. Apparently, the natural competitive microflora prevented growth and thus limited higher concentrations of aflatoxin in maize.     

非脱羧勒克菌wt16对黄曲霉菌生长与产毒的抑制作用

黄曲霉菌及其毒素严重威胁农产品质量安全,本文旨在探寻非脱羧勒克菌对黄曲霉菌及其毒素污染防控效果。从湖北黄陂分离筛选出一株非脱梭勒克菌wt16,将其与黄曲霉菌在液体培养基中共培养后测定非脱羧勒克菌wt16菌株对黄曲霉菌生长及产毒的抑制率。结果表明,在沙氏液体培养基中,非脱羧勒克菌wt16能明显抑制黄曲霉菌的生长及产毒,对其菌丝生长的抑制率为77%~92%,对其产毒的抑制率为90%~96%。通过扫描电子显微镜观察发现,非脱羧勒克菌wt16能改变黄曲霉菌丝的形态,使得黄曲霉菌丝体由规则的球体聚集成不规则形状,单个菌丝会由细长型断裂成小截形态,菌丝表面也变得更为光滑;并且发现wt16在花生粉及未受机械损伤的花生颗粒上对黄曲霉菌的生长及产毒均表现出很强的抑制作用。进一步研究发现,非脱羧勒克菌wt16菌株发酵上清液中含有能抑制黄曲霉毒素合成的有效成分,且该发酵上清液的制备以培养4 d以上为最佳,培养温度为15~40 ℃。     

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.