Impact of volatile compounds generated by essential oils on Aspergillus section Flavi growth parameters and aflatoxin accumulation

BACKGROUND: The advantage of essential oils is their bioactivity in the vapour phase, a characteristic that makes them attractive as possible fumigants for stored grain protection. In this study the antifungal and antiaflatoxigenic effects of the volatile fractions of five essential oils (EOs) were evaluated by vapour contact on Aspergillus section Flavi isolates. RESULTS: In maize meal extract agar the volatile fractions of Pimpinella anisum L. (anise), Pëumus boldus Mol. (boldus), Hedeoma multiflora Benth. (mountain thyme), Lippia turbinata var. integrifolia (Griseb.) (poleo) and Syzygium aromaticum L. (clove) were able to decrease the growth rate and lag phase of aflatoxigenic isolates. Boldus EO showed the best antifungal effect on Aspergillus section Flavi growth rate. In sterilised maize grains, boldus and poleo EOs showed antifungal effects on growth rate and aflatoxin accumulation. The volatile fraction of boldus EO completely inhibited the growth of isolates at water activity (a_w) levels of 0.955, 0.930 and 0.900, while poleo EO showed this effect only at the lower a_w levels (0.930 and 0.900). All aflatoxigenic isolates showed reduced total aflatoxin accumulation in the presence of boldus EO under all a_w conditions. CONCLUSION: These findings clearly indicate that the volatile fraction of boldus EO could be used to control aflatoxigenic fungi in stored maize. Copyright © 2009 Society of Chemical Industry     

Potential of aflatoxin B1 production by Aspergillus flavus strains on commercially important food grains

In this study, we investigated the potential of aflatoxin B₁ (AFB1) production by five Aspergillus flavus strains previously isolated from sorghum grains on cereals (barley, maize, rice, wheat and sorghum), oilseeds (peanuts and sesame) and pulses (greengram and horsegram). Five strains of A. flavus were inoculated on all food grains and incubated at 25 °C for 7 days; AFB1 was extracted and estimated by enzyme‐linked immunosorbent assay. All A. flavus strains produced AFB1 on all food grains ranging from 245.4 to 15 645.2 μg kg^?1. Of the five strains tested, strain Af 003 produced the highest amount of AFB1 on all commodities ranging from 2245.2 to 15 645.2 μg kg^?1. Comparatively, the AFB1 accumulation was high on rice grains ranging from 3125.2 to 15 645.2 μg kg^?1, followed by peanuts ranging from 2206.2 to 12 466.5 μg kg^?1. Less AFB1 accumulation was observed in greengram and sesame seeds ranging from 645.8 to 2245.2 and 245.4 to 2890.6 μg kg^?1, respectively. Our results showed that all food grains tested are susceptible to A. flavus growth and subsequent AFB1 production.     

含水量对玉米粉贮藏期黄曲霉生长及黄曲霉毒素B1积累的影响

目的 研究含水量对玉米粉贮藏过程中黄曲霉生长及黄曲霉毒素B1积累的影响。方法 将初始水分含量10.42%、13.26%、16.35%、19.88%的玉米粉在28 ℃, 相对湿度75%的模拟环境中储藏30 d; 测定贮藏期间玉米粉含水量、黄曲霉生长量及黄曲霉毒素B1的积累量。结果 贮藏期间玉米粉中含水量呈现先上升后下降的变化趋势, 黄曲霉生长量及黄曲霉毒素B1的积累量随贮藏时间的延迟先快速升高, 后趋于平稳。初始含水量较高的玉米粉贮藏期间含水量、黄曲霉生长量及黄曲霉毒素B1含量始终处于较高的水平。相关性分析表明, 玉米粉含水量、黄曲霉生长量和毒素积累量之间均呈极显著的正相关(P<0.01)。结论 含水量是影响玉米粉贮藏过程中霉菌生长和毒素积累的关键因素, 降低玉米粉初始含水量可以有效减轻贮藏期间真菌危害。     

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.     

Phenolic compounds in peanut seeds: Enhanced elicitation by chitosan and effects on growth and aflatoxin B1 production by Aspergillus flavus

Effects of chitosan and Aspergillus flavus to enhance elicitation of phenolic compounds in viable peanut seeds were conducted at two water activity levels. In vitro effects of phenolic acids on A. flavus growth and aflatoxin B₁ production were also studied. Chitosan enhanced elicitation of free phenolic compounds (FPC) at Aw .85 and .95 levels. A. flavus initially decreased and subsequently increased FPC content, but bound phenolic compounds (BPC) decreased during incubation. Chitosan + A. flavus treatment caused an increase in FPC reaching a plateau between 24–48 h at Aw .85 while BPC levels increased over the same period at both Aw levels. Major free and bound phenolic acids detected were p‐coumaric, ferulic and an unknown phenolic acid eluting at a retention time of 22 min. Generally, chitosan significantly enhanced elicitation of free ferulic and p‐coumaric acids and bound p‐coumaric acid at Aw .95. Free unknown phenolic and bound ferulic acids at Aw .85 were enhanced by chitosan. A. flavus caused significant induction of bound p‐coumaric and ferulic acids and free unknown phenol at Aw .85. Chitosan + A. flavus enhanced free p‐coumaric (3 h) and unknown phenolic acids and bound p‐coumaric acid at Aw .95 while bound ferulic acid was enhanced at Aw .85. Chitosan limited A. flavus growth and subsequent aflatoxin production by inducing susceptible tissues to produce more preformed phenolic compounds.

Analysis of liquid cultures of A. flavus revealed that p‐coumaric, ferulic, and vanillic acids and a mixture of these phenolic acids slightly inhibited mycelial growth. Production of aflatoxin B₁ by A. flavus was completely inhibited at 1 mM and 10 mM concentrations of the phenolic acids and their mixture on four days of incubation. Mode of action of phenolic acids is likely on the secondary pathway for aflatoxin B₁ production and not on the primary metabolism for fungal growth.     

Inhibitory effect of Aspergillus niger on the growth of Aspergillus ochraceus and Aspergillus flavus,and on aflatoxin formation

Aspergillus ochraceus and A flavus were grown on synthetic media (SM) supplemented with 50 or 200 ml litre^?1 SM on which A niger had been grown previously ( ‘A niger medium’ = ANM). Controls included SM acidified to pH 6.0 or 4.4, SM diluted with 50 or 200 ml litre^?1 water, and diluted-acidified SM. For both fungi, higher growth inhibition was recorded on ANM-containing SM than in the controls. Aflatoxin formation was markedly inhibited on SM to which 20 ml litre^?1 ANM extract (in methanol/chloroform, 2:1 v) had been added, although the growth of A flavus on that medium was almost the same as that in the control. It is concluded that the inhibitory effect of A niger on the growth of fungi should not be attributed merely to pH reduction, but also, mainly, to metabolites produced by the fungus in the growth medium, even at early stages of its growth.     

Production of aflatoxin B1 and G1 by Aspergillus parasiticus on silica gels.

Silica gels were prepared by acidifying alkaline silicate solutions with phosphoric or tartaric acid. Various combinations of glucose, sucrose, yeast extract, and salts were included in the gels an nutrients. Maximum production of aflatoxins B1 and G1 occurred when silica gel (0.4 to 0.5 cm deep in a petri dish) containing 20% sucrose and 2% yeast extract, and gelled with tartaric acid, was inoculated with approximately 120 to 12000 spores of Aspergillus parasiticus per plate; and plates were incubated at 28 degrees C for 10 days.     

植物提取物抑制玉米中黄曲霉生长及产毒研究

通过液体培养 直接接触法比较植物提取物肉桂醛、柠檬醛和丁香酚对黄曲霉生长及产毒的抑制作用,选取抑制作用最强的肉桂醛应用到玉米中,研究了添加量、玉米水分含量和储藏温度对肉桂醛抑制黄曲霉生长及产毒的影响。结果表明：液体培养基中,肉桂醛、柠檬醛和丁香酚的最低杀菌浓度与各自完全抑制产毒浓度一致,分别为 100、500和500 μl/L。随着添加量的加大,肉桂醛对黄曲霉生长和产毒的抑制率逐渐升高,挥发浓度为96 μl/L时完全抑制黄曲霉生长,对黄曲霉毒素B1的抑制率为97.98％。在玉米水分为14%~40％时,肉桂醛对玉米黄曲霉污染的抑制率随水分含量的升高显著下降,14％时抑制率最高,为88.24%;对黄曲霉毒素B1始终保持较强的抑制作用,14％时产毒抑制率最高,为97.51％。储藏温度在20~37℃时,肉桂醛对黄曲霉污染的抑制率随温度的升高呈现先升高后降低的趋势,28℃黄曲霉污染抑制率最高,为85.67%;对黄曲霉毒素B1抑制率随温度的升高呈现先降低后升高又降低的趋势,20℃时抑制率最高,达到91.00%。     

Influence of Listeria monocytogenes and environmental abiotic factors on growth parameters and aflatoxin B1 production by Aspergillus flavus

Aspergillus flavus growth is influenced by a variety of abiotic factors including water activity (a_W), pH, redox potential, relative humidity, temperature and availability of oxygen. Furthermore, biotic factors such as the presence in the same habitat of different microorganisms can develop ecological interactions between them that are positive, negative or indifferent for fungal growth. The aim of the present work was evaluate the effect of Listeria monocytogenes in interaction with a_W, pH and temperature on the lag phase, growth rate and AFB₁ production of A. flavus. Ecophysiological in vitro studies of A. flavus demonstrate that the co-occurrence with L. monocytogenes in culture medium and abiotic factors (a_W, pH and temperature) influence the growth of the fungal strains and the production of AFB₁. The presence of L. monocytogenes generally decreased fungal growth and increased significantly aflatoxin production. These studies are essential in the prediction of contamination risk of food with these microorganisms or their toxins in interaction with several environmental parameters.     

Effect of γ‐irradiation on aflatoxin B1 production by Aspergillus flavus and chemical composition of three crop seeds

The effect of γ‐irradiation on aflatoxin B₁ production by Aspergillus flavus, and the chemical composition of some different crop seeds were investigated. A. flavus infected seeds behaved differently according to their principal constituents. A. flavus caused an increase in protein and decrease in lipids and carbohydrate contents of wheat, soyabean and fababean seeds. Growth of A. flavus and production of aflatoxin B₁ was inhibited at a dose level of 5 kGy. A. flavus utilizes carbohydrates of seeds for its growth and aflatoxin production. Crops were arranged, in descending order, according to aflatoxin produced in seeds as wheat > soyabean > fababean. There were no changes in chemical constituents of irradiated seeds, such as protein, lipids, and carbohydrates.     

Saccharomyces cerevisiae strains and the reduction of Aspergillus parasiticus growth and aflatoxin B1 production at different interacting environmental conditions,in vitro

The effect of Saccharomyces cerevisiae RC008 and RC016, previously selected based on their aflatoxin B₁ binding ability and beneficial properties, against Aspergillus parasiticus under different interacting environmental conditions was evaluated. Studies concerning the lag phase, growth rate and aflatoxin B₁ production were carried out in vitro under different regimes of a _w (0.95 and 0.99), pH (4 and 6), temperature (25 and 37°C), and oxygen availability (normal and reduced). Both yeast strains showed great antagonistic activity at pH 4, decreasing growth rate compared with the control. The RC008 strain showed the greatest inhibitory activity at all assayed conditions. A. parasiticus produced large amounts of AFB₁ in vitro. A significant decrease of AFB₁ levels in comparison with the control were observed with yeast interaction. Differences between control and treatment values ranged from 130 to 5400?ng?ml^?1. S. cerevisiae RC008 and RC016 could be considered as effective agents in reducing growth and AFB₁ production at different interacting environmental conditions, related to that found in stored feedstuff. The importance of the present work lies in the search for live strains with both probiotic and biocontrol properties able to prolong the safe storage of feedstuff and exert beneficial properties after animal consumption and which could be included in a novel product for animal feed.     

Inhibitory effect of Cynara cardunculus L. extract on aflatoxin B1 production by Aspergillus parasiticus in sesame (Sesamum indicum L.)

Aflatoxin B₁ has been considered as the most potent liver carcinogen for humans. One factor that stimulates the production of aflatoxins in oilseed products, such as sesame seeds and their products is the presence of high levels of fatty acids. This work presents further evidence that sesame is a favorable substrate for aflatoxin B₁ production by Aspergillus parasiticus. Moreover, the use of wild artichoke (Cynara cardunculus L.) extract was examined showing inhibition of aflatoxin B₁ production in both sesame and yeast extract sucrose medium, inoculated with A. parasiticus. More specifically, the inhibition of aflatoxin B₁ production by A. parasiticus in sesame seeds paste was 99.6% and in yeast extract sucrose medium it was 99.4%.     

植物源天然产物对黄曲霉和黄曲霉毒素B_1的抑制作用研究进展

黄曲霉毒素B1(aflatoxin B1,AFB1)是化学物质中致癌性最强的一种,对人和动物的健康具有极大的威胁,尤其是对肝脏的损害。AFB1是谷物和粮油中最为多见的真菌毒素,危害性也最强,长期低剂量的摄入AFB1除了会导致肝脏损伤和诱发肝癌之外,也会损害人体的其他器官。因此,能够有效防护人体免遭AFB1损伤的功能食品和新型保健药物已成为研究重点。近年来,天然产物因其具有生物活性显著、副作用小、多靶点等优点而备受关注,多种植物源天然产物已被报道能够抑制黄曲霉的生长产毒和AFB1的毒性作用。该文主要综述了植物源天然产物对黄曲霉生长产毒和AFB1毒性作用的抑制作用以及其相关抑制机制,为进一步开展AFB1毒性损伤的防护研究工作,开发能降低AFB1对人和动物的毒性损伤的新型保健药物和功能食品提供参考。     

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 γ‐irradiation and maize lipids on the production of aflatoxin B1 by Aspergillus flavus

The effect of γ‐irradiation and maize lipids on aflatoxin B₁ production by Aspergillus flavus artificially inoculated into sterilized maize at reduced water activity (a_w 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 γ‐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 a_w 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.     

Mould infection and aflatoxin contamination of the peanut kernels harvested from spring and fall crops as affected by artificial inoculation of the seeded kernels with Aspergillus flavus and Aspergillus niger

Spring and fall crops of peanut are grown each year in Taiwan. Mould infection and aflatoxin contamination of crops as affected by artificial inoculation of the seeded kernels with conidia of Aspergillus flavus, A niger and a combination of A flavus and A niger (inocula > 10⁵ CFU kernel⁻¹) were determined. Three cultivars, ie Tainan 9, Tainan 11 and Tainan 12, were consecutively grown for fall 1996, spring 1997, fall 1997 and spring 1998 crops with green vegetable pea for rotation. In crops from uninoculated kernels (control), percentages of germination and harvested plants were higher in spring crops than in fall crops. Inoculation with A niger alone or with A flavus resulted in various levels of seed and seedling mortality and lower yields of peanut pods than yields of the other inoculation treatments. When harvested and sized (US No 1), kernels were subjected to examination for mould colonisation and analysis of aflatoxin content. Fairly low percentages were colonised and aflatoxin contents were low or non-detectable. Colonisation and aflatoxin content were independent of artificial inoculation. Average aflatoxin contents in the kernels harvested from all cultivars and crops ranged from 0 to 6.1 µg kg⁻¹. However, the highest levels of aflatoxin content among samples of the four crops were 4.0, 18.2, 9.6 and 36.7 µg kg⁻¹, respectively. © 1999 Society of Chemical Industry