Aflatoxin B1 Synthesis Research Articles

The Potential of Plant-Based Bioactive Compounds on Inhibition of Aflatoxin B1 Biosynthesis and Down-regulation of aflR, aflM and aflP Genes.

The use of plant extracts in pre- and post-harvest disease management of agricultural crops to cope with aflatoxin B1 contamination has shown great promise due to their capability in managing toxins and safe-keeping the quality. We investigated the anti-aflatoxigenic effect of multiple doses of eight plant extracts (Heracleum persicum, Peganum harmala, Crocus sativus, Trachyspermum ammi, Rosmarinus officinalis, Anethum graveolens, Berberis vulgaris, Berberis thunbergii) on Aspergillus flavus via LC-MS and the down-regulatory effect of them on aflR, aflM and aflP genes involved in the aflatoxin B1 biosynthesis pathway using RT-qPCR analyses. Our results showed that H. persicum (4 mg/mL), P. harmala (6 mg/mL) and T. ammi (2 mg/mL) completely stopped the production of aflatoxin B1, without inducing significant changes in A. flavus growth. Furthermore, our findings showed a highly significant correlation between the gene expression and the aflatoxin B1 biosynthesis, such that certain doses of the extracts reduced or blocked the expression of the aflR, aflM and aflP and consequently reduced the synthesis of aflatoxin B1. Interestingly, compared to the regulatory gene (aflR), the down-regulation of expression in the structural genes (aflM and aflP) was more consistent and correlated with the inhibition of aflatoxin B1 production. Overall, this study reveals the anti-aflatoxigenic mechanisms of the selected plant extracts at the gene expression level and provides evidence for their use in plant and crop protection.

Inhibition of aflatoxin B1 biosynthesis and down regulation of aflR and aflB genes in presence of benzimidazole derivatives without impairing the growth of Aspergillus flavus

Aflatoxins are mutagenic secondary metabolites produced by certain ubiquitous saprophytic fungi. These contaminate agricultural crops and pose a serious health threat to humans and livestock all over the world. Benzimidazole and its derivatives are biologically active heterocyclic compounds known for their fungicidal activity. In the present study, second and sixth position substituted benzimidazole derivatives are tested for their antifungal and anti-aflatoxigenic activity. Aflatoxigenic strain of Aspergillus flavus cultured in Yeast extract sucrose (YES) medium as well as in rice in the presence and absence of test compounds. 2-(2-Furyl) benzimidazole (FBD) showed complete inhibition of fungal growth at 50 μg/mL. However, the polar derivatives of FBD viz. 6-NFBD, 6-AFBD, 6-CAFBD, and 6-CFBD did not impair the fungal growth but effectively inhibited aflatoxin B1 biosynthesis. Significant down-regulation of aflR gene involved in regulation and aflB structural gene for aflatoxin B1 biosynthesis was observed in presence of 6-NFBD. These benzimidazole derivatives also showed good anti-aflatoxigenic activity in rice, though the IC50 concentrations in rice were comparatively higher than those in YES medium. This study summarizes the most notable structure-activity relationship (SAR) of 2-(2-Furyl) benzimidazoles for anti-aflatoxigenic and anti-fungal activities. These molecules can be further studied for their applications in industrial fermentation processes vulnerable to mold growth and subsequent aflatoxin B1 synthesis like koji fermentation, cheese production, etc.

Efficacy of different citrus essential oils to inhibit the growth and B1 aflatoxin biosynthesis of Aspergillus flavus.

Food contamination by aflatoxin B1 (AFB1), produced by mycotoxigenic strains of Aspergillus spp., causes severe medical and economic implications. Essential oils (EOs) are mixtures of eco-friendly natural volatile substances. Their ability to inhibit fungal growth has been investigated, while no data are available about their efficacy in inhibition of AFB1 biosynthesis. This study investigates the efficacy of five different citrus EOs to inhibit the growth and AFB1 synthesis of A. flavus through in vitro tests for a future application in food matrices. AFB1 detection was carried out by LC-ESI-TQD analytical approach. Lemon (Citrus limon (L.) Burm. f.), bergamot (Citrus bergamia Risso), and bitter orange (Citrus aurantium L.) EOs were the most effective causing a 97.88%, 97.04%, and 96.43% reduction in mycelial growth, respectively. Sweet orange and mandarin EOs showed the lowest percentage of mycelial growth reduction. Citrus EOs showed different capacity of AFB1 inhibition (lemon > bitter orange > bergamot > sweet orange > mandarin). Our results showed a dose-dependent antifungal activity of lemon, bitter orange, and bergamot EOs which at 2% (v/v) inhibited both mycelium growth and AFB1 genesis of A. flavus. Our results show that EOs' use can be a pivotal key to recovery and reuse of citrus fruit wastes and to be used as eco-friendly fungicides for improvement of food safety. The use of EOs obtained at low cost from the residues of citric industry presents an interesting option for improving the profitability of the agriculture.

Antifungal and antiaflatoxinogenic activities of Carum carvi L., Coriandrum sativum L. seed essential oils and their major terpene component against Aspergillus flavus

With the purpose of searching for biological substances for controlling aflatoxins production in foodstuffs, we assessed the antifungal and antiaflatoxigenic activities of two essential oils (EOs) extracted from Carum carvi L., Coriandrum sativum L. seed and their major terpene component against Aspergillus flavus. The chemical profiles of Carum carvi, and Coriandrum sativum EOs were identified through Gas chromatography mass spectrometry analysis where carvone and linalool were respectively, recorded as the major terpene compound (78.85 and 72.34%). The effects of the EOs (0.1–0.7% (v/v)) and terpenes (100–2000 μg/mL) on the hyphal extension and aflatoxin B1 (AFB1) synthesis of A. flavus were tested by contact assay in Yeast Extract Sucrose medium. Carum carvi EO displayed a higher antimicrobial activity against A. favus than Coriandrum sativum EO with a minimum inhibitory concentration of about 0.4 and 0.7%, respectively. In the presence of 0.3% of EOs, 73.3 and 99.6% inhibition of AFB1 production was recorded, respectively, with Coriandrum sativm and Carum carvi. The antifungal and antiaflatoxigenic effects of carvone were also more significant than those of linalool (p < 0.05) with respectively, 64 and 16% of growth rate inhibition and 77.9 and 0.1% of AFB1 synthesis decrease recorded at 1000 μg/mL. Hence, the antifungal and antiaflatoxigenic activities of the two tested EOs were similar to their main terpene component. In conclusion, seed essential oils proved to be apotential natural source of antifungal and aflatoxin inhibition agent against Aspergillus flavus.

Evaluation of Protective Impact of Algerian Cuminum cyminum L. and Coriandrum sativum L. Essential Oils on Aspergillus flavus Growth and Aflatoxin B1 Production.

Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. The aim of this investigation was to evaluate the inhibition of growth Aspergillus flavus E73 (A. flavus E73) and AFB1 production by Cuminum cyminum L. (C. cyminum L.) and Coriandrum sativum L. (C. sativum L.) essential oils (EOs) as well their antioxidant and phytotoxicity activities. The C. cyminum L. and C. sativum L. EOs were extracted by hydrodistillation. The chemical profile of EOs was identified by GC-MS, antifungal activity was assessed by poisoned food technique and in term Minimal Inhibitory Concentration (MIC) and minimal fungicidal concentration (MFC) and antiaflatoxin effect by broth medium. The antioxidant activity of EOs was determined by DPPH free radical scavenging assay, β-carotene bleaching test and total phenolic content by Folin-Ciocalteu. Phytotoxicity of C. cyminum L. and C. sativum L. EOs were determined for varieties of wheat. The results were analyzed by analysis of variance (one way ANOVA). The GS/MS analysis showed that the major components of C. cyminum L. EO were cuminaldehyde (65.98%), o-cymene (18.40%) and C. sativum L. EO was mainly consisted of linalool (78.86%). The results showed that both the EOs could inhibit the growth of A. flavus E73 in the range of 24.27-84.90% for C. cyminum and 15.09-65.00% for C. sativum. During antiaflatoxin investigation, the oils exhibited noticeable inhibition on dry mycelium weight and synthesis of AFB1 by A. flavus E73. EOs of C. cyminum L. and C. sativum L. revealed complete inhibition of AFB1 at 1.25 and 1.5 mg mL-1, respectively. EOs exhibited inhibitory influence against some fungi. The IC50 values of C. cyminum L. and C. sativum L. EOs were 494.93 and 756.43 μg mL-1, respectively, while, β-carotene/linoleic acid bleaching was 47.68 and 29.29% , respectively. Total phenolic content of C. cyminum L. and C. sativum L. were 10.66 and 6.2 μg mg-1. Additionally, the EOs were non-phytotoxic on the two verities of wheat seeds. The C. cyminum L. and C. sativum L EOs could be good alternative to protect foods.

English

The aim of this study is to explore the impact of Lycium halimifolium Mill leaves extracts on mycotoxin B1 production in vitro, as well as the inhibitor effect of the aflatoxin B1 synthesis by Aspergillus flavus strain. The strain was isolated, identified (gi |146746162 | EF409803.1.) and positively tested for the production of Aflatoxin B1. The antimycotoxinogenesis effect was investigated using L. halimifolium Mill leaves consisting of butanol and ethyl acetate fractions. Indeed, the fractions were tested to stop the secretion of aflatoxin B1 by A. flavus in submerged culture (YES), simultaneously. The growth of the strain was evaluated as biomass response. The antimycotoxinogenesis activity showed that the two fractions: ethyl acetate and butanol of L. halimifolium Mill leaves were able to inhibit significantly the synthesis of mycotoxin AFB1. Thus, the secretion inhibition percentage was calculated in comparison with the control, using HPLC-PDA technique, and estimated at 96.83% for butanolic fraction and 94.99% for ethyl acetate fraction. To verify its correlation with biomass, the growth inhibition percentage was also estimated. The results showed that the dry weight concentration under butanolic and ethyl acetate fractions was 1.6 and 2 g/l, respectively, corresponding to 65.22 and 56.53% of inhibition growth percentage. Key words: Aflatoxin B1, antimycotoxinogenesis activity, high performance liquid chromatography coupled with photodiode array detector (HPLC-PDA), natural extract.

Identification of the Anti-Aflatoxinogenic Activity of Micromeria graeca and Elucidation of Its Molecular Mechanism in Aspergillus flavus

Of all the food-contaminating mycotoxins, aflatoxins, and most notably aflatoxin B1 (AFB1), are found to be the most toxic and economically costly. Green farming is striving to replace fungicides and develop natural preventive strategies to minimize crop contamination by these toxic fungal metabolites. In this study, we demonstrated that an aqueous extract of the medicinal plant Micromeria graeca—known as hyssop—completely inhibits aflatoxin production by Aspergillus flavus without reducing fungal growth. The molecular inhibitory mechanism was explored by analyzing the expression of 61 genes, including 27 aflatoxin biosynthesis cluster genes and 34 secondary metabolism regulatory genes. This analysis revealed a three-fold down-regulation of aflR and aflS encoding the two internal cluster co-activators, resulting in a drastic repression of all aflatoxin biosynthesis genes. Hyssop also targeted fifteen regulatory genes, including veA and mtfA, two major global-regulating transcription factors. The effect of this extract is also linked to a transcriptomic variation of several genes required for the response to oxidative stress such as msnA, srrA, catA, cat2, sod1, mnsod, and stuA. In conclusion, hyssop inhibits AFB1 synthesis at the transcriptomic level. This aqueous extract is a promising natural-based solution to control AFB1 contamination.

In vitro anti-aflatoxigenic effect and mode of action of cinnamaldehyde against aflatoxin B1

Cinnamaldehyde (CIN), a natural extract from Cinnamomum spp., is commonly used for extending the shelf life of selected foods as preservative. In this study, CIN was applied to investigate its effect on aflatoxin B1 (AFB1) biosynthesis of Aspergillus flavus. To elucidate the mechanism of action, the ability of CIN to inhibit fungal growth as well as spore germination of A. flavus was evaluated in vitro. Moreover, the effects of CIN on pellet size, reactive oxygen species formation and lipid peroxidation involved in AFB1 synthesis were furthermore investigated. The results demonstrated that CIN dose-dependently inhibited AFB1 production, which is consistent with the significant reduction of both reactive oxygen species and thiobarbituric acid reactive substances level after CIN treatment for three days. In addition, optical microscope observation showed that CIN could effectively postpone spore germination of A. flavus which is unfavorable for hyphae elongation and pellet formation. However, the trend of AFB1 reduction in YES broth was not exactly consistent with the growth inhibition of A. flavus strain expressed as mycelia dry weight. When fungus was incubated with high concentration of CIN, the degradation rate of up to 14.8% of AFB1 could be achieved on the 5th day. The present study suggested that CIN suppression of AFB1 contamination is due to pleiotropic effects that control spore germination, oxidative stress alleviation of fungal cells and aflatoxin detoxification. These results further showed that CIN could be an alternative to chemical fungicides to prevent A. flavus and AFB1 contamination during grain storage and in the field.

Effect of Carum copticum essential oil on growth and aflatoxin formation by Aspergillus strains

The objectives of this study were to determine the antiaflatoxin B1 activity in vitro of the essential oil (EO) extracted from the seeds of Carum copticum and to evaluate its antifungal activity in vivo as a potential food preservative. The C. copticum EO exhibited noticeable inhibition on dry mycelium and synthesis of aflatoxin B1 (AFB1) by Aspergillus flavus, completely inhibiting AFB1 production at 4 μL/mL. C. copticum EOs showed the lowest percentages of decayed cherry tomatoes for all fungi compared with the control at 100 μL/mL with values of 5.01 ± 67% for A. flavus and 5.98 ± 54% for Aspergillus niger. The results indicated that the percentage of infected fruits is significantly (p < 0.01) reduced by the EO at 16°C for 30 days. In this case, the oil at 100 μL/mL concentration showed the highest inhibition of fungal infection with a value of 80.45% compared with the control. Thus, the EO of dill could be used to control food spoilage and as a potential source of food preservative.

Perillaldehyde, a potential preservative agent in foods: Assessment of antifungal activity against microbial spoilage of cherry tomatoes

The application of natural preservatives has recently become a very attractive method of controlling postharvest decay. The objective of this study was to test the antifungal properties of perillaldehyde (PAE) in vitro and in vivo against four spoilage fungi of cherry tomatoes (Solanum lycopersicum L.): Aspergillus flavus, Aspergillus oryzae, Aspergillus niger and Alternaria alternata. The effect of PAE was evaluated on the mycelial growth and spore germination in the four tested fungi, as well as mycelium weight and AFB1 content in A. flavus. Effect of PAE in the conservation of cherry tomatoes was also assessed. The mycelial growth of the tested fungi was totally inhibited at 0.5 and 0.08 mL/L PAE in the air at contact and vapor conditions, respectively. Spore germination was inhibited in a dose-dependent during exposure to PAE. PAE inhibited synthesis of aflatoxin B1 by A. flavus at 0.4 mL/L. In vivo experiments indicated that all applied concentrations of PAE inhibited the four tested fungi and a broad spectrum of fungal microbiota growth on cherry tomatoes when compared with the control groups. Thus, PAE would be a promising bioactive compound for use as an alternative natural preservative to control the common postharvest spoilage of cherry tomatoes.

Zanthoxylum molle Rehd. essential oil as a potential natural preservative in management of Aspergillus flavus

Essential oils are attracting attention owing to their potential as natural antimicrobial agents, and therefore, they find many applications in the food and pharmaceutical industries. This investigation aimed to evaluate the antifungal activity of Zanthoxylum molle essential oil (ZMEO) against Aspergillus flavus, a food contaminant. Gas chromatography–mass spectrometry detected 51 compounds representing 95.72% of the ZMEO. 2-Undecanone (26.81%), limonene (20.12%), and terpinen-4-ol (13.09%) were the main components. The mycelial growth of A. flavus was totally inhibited at concentrations of 8μL/mL and 1μL/mL ZMEO in the air at contact and vapor conditions, respectively. The ZMEO also had an inhibitory effect on spore germination in A. flavus proportional to its concentration. Moreover, the ZMEO presented evident inhibition on dry mycelium weight and synthesis of aflatoxin B1 (AFB1) by A. flavus, completely restraining AFB1 production at 6μL/mL. This study explores the possible mechanism of the ZMEO against A. flavus by transmission electron microscopy based on changes in the cell ultrastructure. Based on the promising antifungal activity of ZMEO, the effect of the oil on resistance to decay development in cherry tomatoes (Lycopersicon esculentum) was studied in vivo by exposing inoculated and control fruit groups to essential oil vapor at 0.4μL/mL air concentration. The results showed that ZMEO can potentially be used as an antifungal agent for preservation purposes in the food and pharmaceutical industries.

Analysis of the contrast between natural occurrence of toxigenic Aspergilli of the Flavi section and aflatoxin B1 in cassava

Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin produced by Aspergilli of the section Flavi that may contaminate food, in the field or during storage. Cassava represents an important staple food in sub-Saharan Africa. The analysis of aflatoxigenic fungi in 36 cassava samples obtained from producers in Benin indicated that 40% were contaminated by Aspergilli of the section Flavi. Upon morphological and molecular characterization of the 20 isolates, 16 belonged to Aspergillus flavus, 2 to Aspergillus parvisclerotigenus and 2 to Aspergillus novoparasiticus. This is the first time that this latter species is isolated from food. Although most of these isolates were toxigenic on synthetic media, no AFB1 contamination was observed in these cassava samples. In order to determine the action of cassava on AFB1 synthesis, a highly toxigenic strain of A. flavus, was inoculated onto fresh cassava and despite a rapid development, no AFB1 was produced. The anti-aflatoxin property was observed with cassava from different geographical origins and on other aflatoxigenic strains of the section Flavi, but it was lost after heating, sun drying and freezing.Our data suggest that fresh cassava is safe regarding AFB1 contamination, however, processing may alter its ability to block toxinogenesis leading to secondary contamination.

Control of Aspergillus flavus and aflatoxin production with spices in culture medium and rice

Cinnamon, cardamom, star anise and clove were studied for their effect on growth of Aspergillus flavus and aflatoxin B1 (AFB1) synthesis. The experiments were carried out in yeast extract sucrose culture broth as well as in rice supplemented with spices. AFB1 produced was analysed qualitatively and quantitatively using thin layer chromatography and high performance liquid chromatography, respectively. At a concentration of 10 mg/ml, cardamom and star anise did not exhibit any antifungal or anti-aflatoxigenic activity in culture broth, whereas cinnamon and clove inhibited A. flavus growth completely. The minimum inhibitory concentrations of cinnamon and clove were 4 and 2 mg/ml, respectively. Concentrations of cinnamon and clove below their minimum inhibitory concentrations showed enhanced fungal growth, while AFB1 synthesis was reduced. Clove inhibited the synthesis of AFB1 significantly up to 99% at concentrations ≥1.0 mg/ml. The spices also inhibited AFB1 synthesis in rice at 5 mg/g, although fungal growth was not inhibited. Clove and cinnamon inhibited AFB1 synthesis significantly up to 99 and 92%, respectively, and star anise and cardamom by 41 and 23%, respectively. The results of this study suggest the use of whole spices rather than their essential oils for controlling fungal and mycotoxin contamination in food grains.

Investigations on the Antifungal Effect of Nerol against Aspergillus flavus Causing Food Spoilage

The antifungal efficacy of nerol (NEL) has been proved against Aspergillus flavus by using in vitro and in vivo tests. The mycelial growth of A. flavus was completely inhibited at concentrations of 0.8 μL/mL and 0.1 μL/mL NEL in the air at contact and vapor conditions, respectively. The NEL also had an evident inhibitory effect on spore germination in A. flavus along with NEL concentration as well as time-dependent kinetic inhibition. The NEL presented noticeable inhibition on dry mycelium weight and synthesis of aflatoxin B1 (AFB1) by A. flavus, totally restraining AFB1 production at 0.6 μL/mL. In real food system, the efficacy of the NEL on resistance to decay development in cherry tomatoes was investigated in vivo by exposing inoculated and control fruit groups to NEL vapor at different concentration. NEL vapors at 0.1 μL/mL air concentration significantly reduced artificially contaminated A. flavus and a broad spectrum of fungal microbiota. Results obtained from presented study showed that the NEL had a great antifungal activity and could be considered as a benefit and safe tool to control food spoilage.

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 6μl/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.

Evaluation of chemically characterised essential oils of Coleus aromaticus, Hyptis suaveolens and Ageratum conyzoides against storage fungi and aflatoxin contamination of food commodities

SummaryThe result of the present investigation explores the efficacy of chemically characterised essential oils (EOs) of Coleus aromaticus, Hyptis suaveolens and Ageratum conyzoides as antifungal and antiaflatoxigenic agent against some storage fungi and the toxigenic strain of Aspergillus flavus (Saktiman 3NSt). Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of these EOs were also determined against the toxigenic strain of A. flavus (Saktiman 3NSt). The EO from C. aromaticus was found to be most effective exhibiting MIC and MFC at 0.1μL mL−1. The EOs also completely checked aflatoxin B1 synthesis in concentration‐dependent manner. In addition, fumigation of stored wheat samples with EOs exhibited remarkable protection (&gt;80%) from fungal infestation showing their efficacy during in vivo storage conditions. Based on the results of the present investigation, the EOs of C. aromaticus, H. suaveolens and A. conyzoides may be recommended as novel plant‐based antifungal and aflatoxin B1 suppressor over the synthetic preservatives.

Management options, technologies and strategies for minimised mycotoxin contamination of rice

Ninety percent of the world's rice is produced and consumed in Asia. Millions of rice producers are resource-poor farmers with a rice area of less than one hectare. Yield increase and the introduction of double-cropping systems have ensured that rice production has kept up with an increasing demand. However, the increased quantities of grain and the second harvest, which is often in the wet season, have increased the problems in traditional postharvest systems. This can be particularly severe in the humid tropics, where post-harvest losses occur because of outdated management practices and technologies, and delays in post-harvest operations. Fungal infestation of rice grains can lead to discoloration, which results in price reductions in most markets. This can also result in rice being contaminated with mycotoxins, which is less visible to consumers. Contamination with ochratoxins, aflatoxins, and other mycotoxins have occasionally been reported in the literature. In the past, this was not seen as a significant problem and the focus was on other commodities such as maize and peanuts. However, recent studies and a massive recall of food products in Japan in September 2008, including sake, shoshu, and rice crackers made from imported rice from China and Vietnam, which were tested positive for aflatoxins, and also for pesticide residues, have renewed interest in looking at mycotoxin problems in rice. Exploratory studies in the Philippines compared best practice post-harvest management with the traditional management practices often used by smallholder farmers and small processors. It was concluded that synthesis of aflatoxin B1 is very likely in suboptimal post-harvest systems, with levels far above legislative limits. This indicates that there might be a considerable mycotoxin problem in rice from smallholder post-harvest operations. Improved post-harvest management options and technologies are available for diversified small-scale post-harvest systems. Small-scale combine harvesters, affordable and simple mechanical dryers and hermetic storage systems can help to avoid delays in the post-harvest chain and thus reduce mycotoxin contamination of rice. The development of strategies to scale out these improved practices and technologies to a large number of smallholder farmers will continue to be the main challenge.

Inhibitory Effects of Akacid®plus on Growth and Aflatoxin Production by Aspergillus parasiticus

The effects of Akacid plus, a novel guanidine-based polymer first introduced as a biocidal and disinfectant agent were studied on Aspergillus parasiticus growth and its aflatoxin (AF) productivity. The fungus was cultured on yeast extract-sucrose (YES) broth in presence of various twofold serial dilutions of 25% Akacid plus (1.5-96 microL/50 mL medium) and then incubated in shaking condition with 150 rev./min at 28 degrees C for 96 h. Based on obtained results, Akacid plus was found to significantly inhibit both growth and aflatoxin B1 (AFB1) synthesis in very low concentrations in a dose-dependent manner. Fungal growth inhibition was determined in the range of 9.6-99.6% in mycelia exposed to the total concentration range of 1.5-48 microL. A final concentration of 96 microL was necessary to completely inhibit the growth of fungus. Under similar conditions, AFB1 synthesis was found to be strongly inhibited by 8.1-98.0% in presence of 1.5-24 microL Akacid plus with a maximum of 100% by 48 microL concentration. With respect to the unique physico-chemical properties of Akacid plus, its marked inhibitory effects on A. parasiticus growth and its AFB1 synthesis shown for the first time in this study make it a promising candidate for application in prevention programmes of AF contamination of susceptible crops.

Fungal degradation of aflatoxin B1.

A number of fungal cultures were screened to select an organism suitable to be used in the detoxification of aflatoxin B1. They were co-cultured in Czapek-Dox-Casamino acid medium with aflatoxin B1 producing Aspergillus flavus. Several fungal cultures were found to prevent synthesis of aflatoxin B1 in liquid culture medium. Among these Phoma sp., Mucor sp., Trichoderma harzianum, Trichoderma sp. 639, Rhizopus sp. 663, Rhizopus sp. 710, Rhizopus sp. 668, Alternaria sp. and some strains belonging to the Sporotrichum group (ADA IV B14(a), ADA SF VI BF (9), strain 720) could inhibit aflatoxin synthesis by > or =90%. A few fungi, namely ADA IV B1, ADA F1, ADA F8, also belonging to the Sporotrichum group, were less efficient than the Phoma sp. The Cladosporium sp. and A. terreus sp. were by far the least efficient, registering <10% inhibition. The cultures which prevent aflatoxin biosynthesis are also capable of degrading the preformed toxin. Among these, Phoma sp. was the most efficient destroying about 99% of aflatoxin B1. The cell free extract of Phoma sp. destroyed nearly 50 microg aflatoxin B1 100 ml(-1) culture medium (90% of the added toxin), and this was more effective than its own culture filtrate over 5 days incubation at 28+/-2 degrees C. The degradation was gradual: 35% at 24 h, 58% at 48 h, 65% at 72 h, 85% at 96 h and 90% at 120 h. The possibility of a heat stable enzymatic activity in the cell free extract of Phoma is proposed.

Production of aflatoxin byAspergillus parasiticus and its control

The aim of the present work was to investigate the production of aflatoxin byAspergillus parasiticus and to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from aflatoxins.Bacillus megatertum andB cereus were suitable for microbiological assay of aflatoxins. Czapek's-Dox medium was found a suitable medium for isolation of fungi from food samples. The optimal pH for the growth ofA. parasiticus and its productivity of aflatoxin B1 was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production (NH4)2HPO4 (1.55 gL(-1)) and NaNO2 (1.6 gL(-1)) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B1 production. Zn(2+) and Co(2+) supported significantly both fungal growth, as well as, aflatoxin B1 production at the different tested concentrations. Zn(2+) was effective when added toA. parasiticus growth medium at the first two days of the culture age. The other tested metal ions expressed variable effects depending on the type of ion and its concentration. Water activity (aw) was an important factor controlling the growth ofA. parasiticus and toxin production. The minimum aw for the fungal growth was 0.8 on both coffee beans and rice grains, while aw of 0.70 caused complete inhibition for the growth and aflatoxin B1 production. H2O2 is a potent inhibitor for growth ofA. parasiticus and its productivity of toxins. NaHCO3 and C6H5COONa converted aflatoxin B1 to water-soluble form which returned to aflatoxin B1 by acidity. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B1 production. Stearic acid supported the fungal growth and decreased the productivity of AFB1 gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B1 production. Vitamins C and D2 were also repressive particularly for aflatoxin production The present study included studying the activities of some enzymes in relation to aflatoxin production during 20-days ofA. parasiticus age in 2-days intervals. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B1 production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B1 synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were associated with the increase of aflatoxin B1 production. All the tested enzymes as well as aflatoxin B1 production were inhibited by either catechol or phenol.