Microbial detoxification of aflatoxin

Yeasts, molds, bacteria, actinomycetes, algae, and fungal spores were screened for their ability to degrade aflatoxin. Some molds and mold spores partially transformed aflatoxin B(1) to new fluorescing compounds. Only one of the bacteria, Flavobacterium (aurantiacum?) NRRL B-184, removed aflatoxin from solution. Both growing and resting cells of B-184 took up toxin irreversibly. Toxin-contaminated milk, oil, peanut butter, peanuts, and corn were completely detoxified, and contaminated soybean was partially detoxified by addition of B-184. Duckling assays showed that detoxification of aflatoxin solutions by B-184 was complete, with no new toxic products being formed.     

Aflatoxin M1 removal from aqueous solutions by Flavobacterium aurantiacum

In liquid cultures growing and stationary phase cells ofFlavobacterium aurantiacum NRRL B-184 eliminated aflatoxin M₁. Toxin concentrations of 15µg/ml and 37.5µg/ml interfered with bacterial growth, and at the higher level 4.4µg M₁ was removed from the growth medium by a milligram (dry weight) of bacteria. Toxin was completely removed from the liquid medium by incubating 5 × 10¹⁰ resting cells per milliliter with 8µg/ml of aflatoxin M₁ for 4 h. Attempted recovery of M₁ from cells following incubation of the bacteria with the toxin demonstrated that the M₁ was essentially nonextractable. Bacterial cells also removed aflatoxin M₁ from toxin-contaminated milk.     

Mechanism of removal of aflatoxin B1 by a resistant bacterium

A bacterium resistant to aflatoxin B1 isolated from the soil was shown to remove a maximum 26% of toxin at 3 microg ml(-1). A comparison of spectrophotometric and radioisotope methods showed that a maximum of 48 hr was sufficient to remove the toxin. Radioisotope analysis showed that the radioactivity decreased in the chloroform phase while it increased in the aqueous phase during the time course of the experiment. An analysis of the supernatant of culture medium showed that the bacterium had converted aflatoxin B1 to water soluble compounds with lambda(max) of 338 and 374.     

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.     

Tightly bound binary toxin in the cell wall of Bacillus sphaericus

We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.     

Conditions for induction of bacteriophage from lysogenic Bacillus megaterium with aflatoxin B1.

The present study was conducted to determine whether or not aflatoxin B1 was an effective inducing agent for lysogenic bacteria and to characterize some of the parameters involved in induction. A lysogenic strain of Bacillus megaterium (NRRL-B-3695) and an indicator strain of this species (NRRL-B-3694) were used. Cultures of the lysogenic strain were incubated for various periods of time in the presence of aflatoxin B1. Plaque-forming units as well as colony-forming units were then determined. Results of the present study indicated that bacteriophage lysogenizing B. megaterium could be induced with aflatoxin B1. The optimum concentration for induction was 25 micrograms of toxin per ml of early-log-phase culture. Evidence suggested that: (i) higher concentrations of aflatoxin B1 formed hydrophobic complexes which would not efficiently induce B. megaterium; (ii) the toxic effect of aflatoxin B1 severely limited the number of cells which could be induced prior to killing action of the toxin; and (iii) concentrations less than 25 micrograms of aflatoxin B1 per ml were not efficient inducers of bacteriophage production nor did they demonstrate the toxic effect observed at higher concentrations.     

Conditions for induction of bacteriophage from lysogenic Bacillus megaterium with aflatoxin B1.

The present study was conducted to determine whether or not aflatoxin B1 was an effective inducing agent for lysogenic bacteria and to characterize some of the parameters involved in induction. A lysogenic strain of Bacillus megaterium (NRRL-B-3695) and an indicator strain of this species (NRRL-B-3694) were used. Cultures of the lysogenic strain were incubated for various periods of time in the presence of aflatoxin B1. Plaque-forming units as well as colony-forming units were then determined. Results of the present study indicated that bacteriophage lysogenizing B. megaterium could be induced with aflatoxin B1. The optimum concentration for induction was 25 micrograms of toxin per ml of early-log-phase culture. Evidence suggested that: (i) higher concentrations of aflatoxin B1 formed hydrophobic complexes which would not efficiently induce B. megaterium; (ii) the toxic effect of aflatoxin B1 severely limited the number of cells which could be induced prior to killing action of the toxin; and (iii) concentrations less than 25 micrograms of aflatoxin B1 per ml were not efficient inducers of bacteriophage production nor did they demonstrate the toxic effect observed at higher concentrations.     

Tightly Bound Binary Toxin in the Cell Wall of Bacillus sphaericus

We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.     

Phage-displayed peptides that mimic aflatoxin B1 in serological reactivity

AIMS: To test phage-displayed random peptide libraries as sources of peptides that mimic the binding of aflatoxin B1 to monoclonal antibodies raised against the toxin. METHODS AND RESULTS: For two of the three MAbs tested, clones were obtained by panning, producing phage that bound specifically to MAb 13D1-1D9 (MAb 24; specific for aflatoxins B1 and G1) and MAb 6E12-1E9 (MAb 13; specific for aflatoxins B1, G1 and B2) in ELISA. The amino acid sequences of the binding peptides varied. Those binding to MAb 24 contained the sequence of '...YMD...', and those that bound to MAb 13 contained the dipeptide 'PW'. Mimotope phage was used in a competition ELISA format for assaying aflatoxin concentrations. CONCLUSION: The results show that mimotope preparations are effective substitutes for pure toxin in these ELISA procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: These results should contribute significantly to enhancing the safety and diminishing the costs of aflatoxin assays.     

Some studies of the effects of aflatoxin B1 in vivo and in vitro on nucleic acid synthesis in rat and mouse.

The mouse compared with the rat, is more resistent to the acute toxic action of aflatoxin B1 and is refractory to its hepatocarcinogenic properties. Aflatoxin B1 inhibits DNA synthesis more strongly than RNA synthesis in the rat, and both nucleic acid syntheses more strongly in rat than in the mouse. Mouse hepatic microsomes, like those of the rat, are capable of metabolizing aflatoxin B1 in vitro in the presence of NADPH, to an active form which binds to DNA both in solution and in intact nuclei and also inhibits nuclear RNA synthesis. Non NADPH-dependent binding of aflatoxin B1 to nuclei is not effective in inhibiting RNA polymerase and is largely removed by washing with lipid solvents. Mouse nuclear RNA polymerases particularly Mn 2+ (NH4)2SO4 primed acitivity are more resistant to inhibition in vitro by activated aflatoxin B1 than are the corresponding enzyme activities in rat liver nuclei. This would appear to be due to the bound aflatoxin B1 being less efficient in the case of the mouse nucleus, in inhibiting RNA synthesis. Mouse liver slices exhibit a much lesser degree of inhibition of RNA synthesis by aflatoxin B1 than do rat liver slices. Accompanying this is a lower level of binding of aflatoxin B1 to subcellular particulate fractions in the mouse liver slice compared to the rat, this disparity being most marked in the case of the nuclear fraction. The suggestion is made that the resistance of RNA synthesis in the mouse liver, to aflatoxin B1, and perhaps also resistance to its toxicity, is dependent, not on a lower capacity to activate the toxin, but (a) on a less efficient inhibition of RNA synthesis by nuclear bound toxin, and (b) a detoxifying mechanism at least partially situated in the cytosol fraction.     

A comparative study of the effect of aflatoxin B1 and actinomycin D on HeLa cells

1. The cytotoxic effects of aflatoxin B(1) on HeLa cells were examined and effects of short exposures of the cells to the toxin were found to be reversible. 2. Aflatoxin B(1) inhibited the synthesis of both ribosomal and heterodisperse RNA. It is proposed that the toxin's mechanism of action on ribosomal RNA synthesis is related to its inhibitory effect on the maturation of the 45s-ribosomal-RNA precursor. 3. Protein synthesis is inhibited to a greater extent by aflatoxin B(1) than by actinomycin D. In contrast with actinomycin D, aflatoxin B(1) was shown to disaggregate polyribosomes directly.     

Bioreduction of Pt (IV) from aqueous solution using sulphate-reducing bacteria

The aims of this study were to investigate the role of sulphate-reducing bacteria in facilitating Pt removal from aqueous solutions and to investigate the role of a hydrogenase enzyme in Pt reduction in vitro. To avoid precipitation of Pt as Pt sulphide, a resting (non-growing) mixed culture was used. A pH-dependent rate of Pt removal from aqueous solution was observed, indicating that metal speciation was the main factor for its removal from solution. The maximum initial concentration of Pt(IV) that the cells can effectively remove from solution was 50 mg/l, while the maximum capacity was only 4 mg of Pt per gram of resting biomass. Transmission electron microscopy and energy dispersive X-ray analyses indicated that Pt was being precipitated in the periplasm, a major area of hydrogenase activity in the cells. In vitro investigation of Pt reduction with hydrogen as the electron donor showed that 49% was removed within 1 h when a relatively pure hydrogenase extract was used, 31% was removed with a cell-free soluble extract and 70% removed by live cells.     

Adsorption of ochratoxin A and zearalenone by potential probiotic Saccharomyces cerevisiae strains and its relation with cell wall thickness

Aims: To examine Saccharomyces cerevisae strains with previously reported beneficial properties and aflatoxin B₁ binding capacity, for their ability to remove ochratoxin A (OTA) and zearalenone (ZEA) and to study the relation between cell wall thickness and detoxificant ability of yeast strains. Methods and Results: A mycotoxin binding assay at different toxin concentrations and the effect of gastrointestinal conditions on mycotoxin binding were evaluated. Ultrastructural studies of yeast cells were carried out with transmission electronic microscopy. All tested strains were capable of removing OTA and ZEA. Saccharomyces cerevisiae RC012 and RC016 showed the highest OTA removal percentage, whereas RC009 and RC012 strains showed the highest ZEA removal percentages. The cell diameter/cell wall thickness relation showed a correlation between cell wall amount and mycotoxin removal ability. After exposure to gastrointestinal conditions, a significant increase in mycotoxin binding was observed. Conclusions: All tested Saccharomyces cerevisiae strains were able to remove OTA and ZEA, and physical adsorption would be the main mechanism involved in ochratoxin A and ZEA removal. Gastrointestinal conditions would enhance adsorption and not decrease mycotoxin–adsorbent interactions. Significance and Impact of the Study: Live strains with mycotoxin binding ability and beneficial properties are potential probiotics that could be included in animal feed. Previous and present results suggest that the RC008 and RC016 strains are very promising candidates for functional feed product development.     

Inactivation of aflatoxin B1 by using the synergistic effect of hydrogen peroxide and gamma radiation.

Inactivation of aflatoxin B1 was studied by using gamma radiation and hydrogen peroxide. A 100-krad dose of gamma radiation was sufficient to inactivate 50 micrograms of aflatoxin B1 in the presence of 5% hydrogen peroxide, and 400 krad was required for total degradation of 100 micrograms of aflatoxin in the same system. Degradation of aflatoxin B1 was confirmed by high-pressure liquid chromatographic and thin-layer chromatographic analysis. Ames microsomal mutagenicity test showed loss of aflatoxin activity. This method of detoxification also reduces the toxin levels effectively in artificially contaminated groundnuts.     

Inactivation of aflatoxin B1 by using the synergistic effect of hydrogen peroxide and gamma radiation

Inactivation of aflatoxin B1 was studied by using gamma radiation and hydrogen peroxide. A 100-krad dose of gamma radiation was sufficient to inactivate 50 micrograms of aflatoxin B1 in the presence of 5% hydrogen peroxide, and 400 krad was required for total degradation of 100 micrograms of aflatoxin in the same system. Degradation of aflatoxin B1 was confirmed by high-pressure liquid chromatographic and thin-layer chromatographic analysis. Ames microsomal mutagenicity test showed loss of aflatoxin activity. This method of detoxification also reduces the toxin levels effectively in artificially contaminated groundnuts.     

Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B₁ complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B₁ remained bound. Nonviable bacteria retained the highest amount of aflatoxin B₁. Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B₁ from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B₁ to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B₁. Variation in temperature (4 to 37°C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B₁ released. Binding of aflatoxin B₁ appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions.     

Determination of mycotoxins in bovine milk by liquid chromatography tandem mass spectrometry

Liquid chromatographic/tandem mass spectrometric methods using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for determination of 18 mycotoxins and metabolites-ochratoxin A, zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol (zeranol), beta-zearalanol (taleranol), fumonisin B1, fumonisin B2, T-2 toxin, HT-2 toxin, T-2 triol, diacetoxyscirpenol (DAS), 15-monoacetoxyscirpenol (MAS), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), deepoxy-deoxynivalenol (DOM-1) and aflatoxin M1--in milk. The mycotoxins were extracted and cleaned up simultaneously. Extraction and removal of lipophilic compounds was performed at pH 2 using a two-phase mixture of acetonitrile and hexane. The acetonitrile concentration of the aqueous phase was reduced and the pH was adjusted to 8.5 before clean up by solid phase extraction (SPE) on Oasis HLB. The toxins DON, DOM-1, 3-AcDON, 15-AcDON, ochratoxin A, zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol and beta-zearalanol were detected in negative ion mode after separation on a Hypersil ENV analytical column, while the toxins T-2 toxin, HT-2 toxin, T-2 triol, DAS, MAS, fumonisin B1, fumonisin B2 and aflatoxin M1 were detected in positive ion mode after separation on a Luna C18 column. Two transition products were monitored for each compound. The extraction and SPE conditions were optimised to obtain maximum recovery and minimum signal suppression/enhancement. The detection capabilities related to the transition products of lowest abundance were in the range 0.020-0.15 microg/l. The mean true recoveries were in the range 76-108% at levels of 0.2-10 microg/l.     

Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.     

Differentiation of aflatoxins from territrems.

Three methods were adopted for differentiation of aflatoxins B1 and B2 from territrems A and B. They were as follows. (i) Then-layer chromatography coupled with chemical confirmation. A significant decrease in the Rf value of trifluoroacetic acid-treated aflatoxin B1 developed in chloroform-acetone (85:15, vol/vol) was satisfactory in differentiating this toxin from the other three. (ii) High-pressure liquid chromatography monitored synchronously at two wavelengths, 365 and 335 nm. The ratio derived from this double-wavelengh detection could serve as an indicator of the presence of each toxin. (iii) Velasco's flurotoxin meter method, which is used for the determination of aflatoxins within the range of 0 to 50 ng/ml, was not significantly affected by territrems even when they were present in quantities at the microgram-per-milliliter level.     

In vitro metabolism of the pro-carcinogen aflatoxin B1 by liver preparations of the calf, nurse shark and clearnose skate

1. Liver postmitochondrial supernatant preparations of calf, clearnose skate, and nurse shark were able to metabolize the fungal toxin aflatoxin B1 to various metabolites. 2. Calf liver produced aflatoxin M1 and Q1 as the major chloroform soluble metabolites, with small amounts of aflatoxicol formed during incubation. 3. Liver preparations of the elasmobranchs, however, produced aflatoxicol as the major chloroform soluble metabolite with no other metabolite being detected. 4. The water soluble metabolite profiles for the three species were also quite different with the tris diol adduct being produced to a much greater extent in calf liver preparations. 5. Aflatoxicol production by the elasmobranch liver homogenates was reversible with the skate reconverting a large amount (30%) of aflatoxicol to AFB1. The nurse shark, however, appeared to convert a portion of aflatoxicol to an unknown metabolite more polar than AFB1. 6. Calf liver DNA bound approximately 3 x more 3H-AFB1 than shark liver DNA.