Degradation kinetics of aflatoxin B1 and B2 in solid medium by using pulsed light irradiation.

BACKGROUND: Pulsed light (PL) is a new potential technology to degrade aflatoxin. The objective of this study was to investigate the degradation characters of aflatoxin B1 (AFB1 ) and B2 (AFB2 ) treated under PL irradiation. A kinetic degradation study of AFB1 and AFB2 in solid medium was performed under PL irradiation at different initial concentrations of AFB1 (229.9, 30.7 and 17.8 µg kg-1 ) and AFB2 (248.2, 32.2 and 19.5 µg kg-1 ) and irradiation intensities (2.86, 1.60 and 0.93 W cm-2 ) of PL. A second-order reaction model was applied to describe degradation of AFB1 and AFB2 . RESULTS: The results showed that the degradation of AFB1 and AFB2 followed the second-order reaction kinetic model well (R2  > 0.97). The degradation rate was proportional to the intensities of PL irradiation and the initial concentrations of aflatoxins. CONCLUSION: It is concluded that the degradation of AFB1 and AFB2 with the use of PL could be accurately described using the second-order reaction kinetic model. © 2018 Society of Chemical Industry.

Effect of UV irradiation on aflatoxin reduction: a cytotoxicity evaluation study using human hepatoma cell line.

In this proof-of-concept study, the efficacy of a medium-pressure UV (MPUV) lamp source to reduce the concentrations of aflatoxin B1, aflatoxin B2, and aflatoxin G1 (AFB1, AFB2, and AFG1) in pure water is investigated. Irradiation experiments were conducted using a collimated beam system operating between 200 to 360 nm. The optical absorbance of the solution and the irradiance of the lamp are considered in calculating the average fluence rate. Based on these factors, the UV dose was quantified as a product of average fluence rate and treatment time. Known concentrations of aflatoxins were spiked in water and irradiated at UV doses ranging from 0, 1.22, 2.44, 3.66, and 4.88 J cm-2. The concentration of aflatoxins was determined by HPLC with fluorescence detection. LC-MS/MS product ion scans were used to identify and semi-quantify degraded products of AFB1, AFB2, and AFG1. It was observed that UV irradiation significantly reduced aflatoxins in pure water (p < 0.05). Irradiation doses of 4.88 J cm-2 reduced concentrations 67.22% for AFG1, 29.77% for AFB2, and 98.25% for AFB1 (p < 0.05). Using this technique, an overall reduction of total aflatoxin content of ≈95% (p < 0.05) was achieved. We hypothesize that the formation of ˙OH radicals initiated by UV light may have caused photolysis of AFB1, AFB2, and AFG1 molecules. In cell culture studies, our results demonstrated that the increase of UV dosage decreased the aflatoxin-induced cytotoxicity in HepG2 cells. Therefore, our research finding suggests that UV irradiation can be used as an effective technique for the reduction of aflatoxins.

Kinetics of aflatoxin degradation during peanut roasting.

This study investigated aflatoxin degradation during peanut roasting. First, peanuts contaminated with three initial aflatoxin concentrations (35, 332 and 695µg/kg) were roasted at 180°C for up to 20min. The percentage of aflatoxin degradation after 20min were 55, 64 and 81% for peanuts contaminated with aflatoxin at 35, 332 and 695µg/kg, respectively. This difference was statistically significant (p<0.05), showing that initial concentration influences aflatoxin reduction. Thereafter, peanut samples contaminated with an initial aflatoxin concentration of 85µg/kg were roasted at 160, 180 and 200°C for 5, 10, 15, 20 and 25min, then residual concentrations of aflatoxin were determined. Roasting at 160, 180 and 200°C resulted in an aflatoxin reduction of 61.6, 83.6 and 89.7%, respectively. This study has provided quantitative data reinforcing the fact that roasting alone is not enough to control aflatoxins in peanuts.

Mycotoxin contamination of animal feedingstuff: detoxification by gamma-irradiation and reduction of aflatoxins and ochratoxin A concentrations.

Mycotoxins are fungal secondary metabolites identified in many agricultural products screened for toxigenic moulds. They have been reported to be carcinogenic, teratogenic, tremorogenic, haemorrhagic and dermatitic to a wide range of organisms. With the increasing stringent regulations for mycotoxins imposed by importing countries such as those of the European Union, many cereals that are not safe for human consumption are used in formulations intended for animal feed. Gamma-rays are reported in the scientific literature to destroy ochratoxin A and aflatoxin in food crops and feed. The present study provides preliminary data for establishing the effect of dose of gamma-irradiation, ranging from 0 to 15 kGy, on aflatoxins and ochratoxin A reduction in commercial animal feed. The mycotoxin levels were determined by means of immunoaffinity clean-up (IAC) and HPLC with fluorescence detection (HPLC-FLD). The maximum reductions found at 15 kGy were 23.9%, 18.2%, 11.0%, 21.1% and 13.6% for ochratoxin A, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2, respectively. Results showed that the gamma-rays even at 15 kGy were not effective in the complete destruction of ochratoxin A and aflatoxins in the tested feed.

Mold and aflatoxin reduction by gamma radiation of packed hot peppers and their evolution during storage.

The effect of gamma radiation on moisture content, total mold counts, Aspergillus counts, and aflatoxins of three hot pepper hybrids (Sky Red, Maha, and Wonder King) was investigated. Whole dried peppers packed in polyethylene bags were gamma irradiated at 0 (control), 2, 4, and 6 kGy and stored at 25°C for 90 days. Gamma radiation proved to be effective in reducing total mold and Aspergillus counts in a dose-dependent relationship. Total mold counts in irradiated peppers immediately after treatments were significantly lowered compared with those in nonirradiated samples, achieving 90 and 99% reduction at 2- and 4-kGy doses, respectively. Aspergillus counts were significantly reduced, by 93 and 97%, immediately after irradiation at doses of 2 and 4 kGy, respectively. A radiation dose of 6 kGy completely eliminated the population of total molds and Aspergillus fungi. The evolution of total molds in control and irradiated samples indicated no further fungal proliferation during 3 months of storage at 25°C. Aflatoxin levels were slightly affected by radiation doses of 2 and 4 kGy and showed a nonsignificant reduction of 6% at the highest radiation dose of 6 kGy. The distinct effectiveness of gamma radiation in molds and aflatoxins can be explained by the target theory of food irradiation, which states that the likelihood of a microorganism or a molecule being inactivated by gamma rays increases as its size increases.

Evaluation of fungal burden and aflatoxin presence in packed medicinal plants treated by gamma radiation.

This study was developed to evaluate the fungal burden, toxigenic molds, and mycotoxin contamination and to verify the effects of gamma radiation in four kinds of medicinal plants stored before and after 30 days of irradiation treatment. Eighty samples of medicinal plants (Peumus boldus, Camellia sinensis, Maytenus ilicifolia, and Cassia angustifolia) purchased from drugstores, wholesale, and open-air markets in São Paulo city, Brazil, were analyzed. The samples were treated using a (60)Co gamma ray source (Gammacell) with doses of 5 and 10 kGy. Nonirradiated samples were used as controls of fungal isolates. For enumeration of fungi on medicinal plants, serial dilutions of the samples were plated in duplicate onto dichloran 18% glycerol agar. The control samples revealed a high burden of molds, including toxigenic fungi. The process of gamma radiation was effective in reducing the number of CFU per gram in all irradiated samples of medicinal plants after 30 days of storage, using a dose of 10 kGy and maintaining samples in a protective package. No aflatoxins were detected. Gamma radiation treatment can be used as an effective method for preventing fungal deterioration of medicinal plants subject to long-term storage.

Correlation and classification of single kernel fluorescence hyperspectral data with aflatoxin concentration in corn kernels inoculated with Aspergillus flavus spores.

The objective of this study was to examine the relationship between fluorescence emissions of corn kernels inoculated with Aspergillus flavus and aflatoxin contamination levels within the kernels. Aflatoxin contamination in corn has been a long-standing problem plaguing the grain industry with potentially devastating consequences to corn growers. In this study, aflatoxin-contaminated corn kernels were produced through artificial inoculation of corn ears in the field with toxigenic A. flavus spores. The kernel fluorescence emission data were taken with a fluorescence hyperspectral imaging system when corn kernels were excited with ultraviolet light. Raw fluorescence image data were preprocessed and regions of interest in each image were created for all kernels. The regions of interest were used to extract spectral signatures and statistical information. The aflatoxin contamination level of single corn kernels was then chemically measured using affinity column chromatography. A fluorescence peak shift phenomenon was noted among different groups of kernels with different aflatoxin contamination levels. The fluorescence peak shift was found to move more toward the longer wavelength in the blue region for the highly contaminated kernels and toward the shorter wavelengths for the clean kernels. Highly contaminated kernels were also found to have a lower fluorescence peak magnitude compared with the less contaminated kernels. It was also noted that a general negative correlation exists between measured aflatoxin and the fluorescence image bands in the blue and green regions. The correlation coefficients of determination, r(2), was 0.72 for the multiple linear regression model. The multivariate analysis of variance found that the fluorescence means of four aflatoxin groups, <1, 1-20, 20-100, and >or=100 ng g(-1) (parts per billion), were significantly different from each other at the 0.01 level of alpha. Classification accuracy under a two-class schema ranged from 0.84 to 0.91 when a threshold of either 20 or 100 ng g(-1) was used. Overall, the results indicate that fluorescence hyperspectral imaging may be applicable in estimating aflatoxin content in individual corn kernels.

Effect of gamma irradiation on elimination of aflatoxins produced by apple mycoflora in apple fruits.

The influence of ionizing radiation on the growth of apple mycoflora and the aflatoxins production was investigated. Four strains of fungal isolates, Aspergillus flavus, A. fumigatuis, A. niger, Penicillium expansum, were used. Four doses of gamma radiation were applied (0.5, 1.0, 1.5 and 2.0 KGy). The elimination of aflatoxins under selected conditions was detected by using HPLC techniques. Doses of 0.5 KGy stimulated the production of aflatoxins by all fungi tested while 1.0-2.0 KGy reduced the aflatoxins production. The studies were also carried out to investigate the effect of pre- and post-inoculation of both irradiated and non-irradiated fungal spores on irradiated and non-irradiated apples. The results showed the necessity of irradiation of fresh apple fruits followed by good preservation conditions. The results showed also that the effect of radiation is influences on the apple mycoflora producing aflatoxins and not the apple itself.

[Aflatoxins in food: tests of decontamination of peanut cakes by ionizing treatment]. / Aflatoxines dans les aliments: essais de décontamination des tourteaux d'arachide par traitement ionisant.

The efficacy of ionising treatment for decontaminating peanut cakes was tested. The influence of cakes water content and the effect of ionisation dose rate were studied. The results obtained after a reverse phase liquid chromatographic determination of B1, B2, G1 and G2 aflatoxins have revealed an important contamination of the peanut cakes (up to 1000 ppb of total aflatoxin's contents). After ionising treatment at 25 kGy, the aflatoxins degradation in peanut cake's was less important in dried samples (about 5-10% at 0.55 water activity: aw) than in the humid ones (40-60% degradation at 0.95 water activity). At this dose, any indicative difference of the degradation rate of aflatoxins, with regard to the ionising process was observed. The efficacy of ionising treatment for decontaminating peanut cakes could probably be improved, however the economic interest of such process as alternative of the treatment with ammonia is questionable.

Enzymatic and non-enzymatic formation of free radicals from aflatoxin B1.

Formation of free radicals from aflatoxin B1 was demonstrated in systems of a) NADPH/microsome, b) H2O2/peroxidase, c) UV irradiation, d) silver oxide oxidation and e) alkaline dimethyl sulfoxide. In a) to c), radical spots were detected in thin-layer chromatograms. Among several known metabolites of aflatoxin B1, aflatoxin M1 was active in generation of free radical. These results suggest a possible role of free radical metabolites in the cytotoxicity of this hepatocarcinogen.

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.

[Photolysis of aflatoxin B1 in the presence of pyridine N-oxide]. / Fotoliz aflatoksina B1 v prisutstvii N-okisi piridina.

Aflatoxin B1 (AFB1) photolysis in presence of pyridine N-oxide was studied. It was established that AFB1 ethanol solution irradiated with UV-light lambda = 362 nm demonstrated an increase of the fluorescence as a result of appearance of AFB1 product which was formed by pyridine N-oxide oxygen binding to AFB1 molecules in position 2,3.

Comparación de los efectos provocados por el calor y la irradiación sobre Aspergillus parasiticus / Comparison of effects resulting from the application of physical treatments to Aspergillus parasiticus

El propósito de este trabajo fue estudiar la inhibición de la produción de aflatoxinas asociadas al crecimiento del hongo Aspergillus parasiticus NRRL 2999, mediante el empleo de radiaciones ionizantes. El hongo fue cultivado en arroz de tipo comercial en condiciones de temperatura y humedad ideales para la producción de toxinas. Los cultivos así obtenidos fueron irradiados con dosis de 1,5 kGy* (2D10) al cabo de distintos tiempos de desarrollo, observándose que la máxima inhibición de la producción de toxinas se alcanzaba al irradiar cultivos de 20 horas de desarrollo. Asimismo, se estudió el efecto del calor y su combinación con la irradiación sobre la producción de aflatoxinas, la cual fue seguida a lo largo de 11 días de desarrollo de los cultivos. El contenido de toxinas de los distintos cultivos se dosó por dilución a estinción en cromatografía de capa delgada. Los resultados obtenidos mostraron que, tanto un calentamiento de 15 min a 55-C, como la irradiación de los cultivos con una dosis de 1,5kGy, disminuyen el contenido de toxinas, siendo este último processo el más efectivo. Más aún, la combinación de estos dos agentes físicos, irradiando los cultivos inmediatamente después de ser sometidos al calentamiento, reduce los niveles de toxinas por debajo de los límites de detección sugeridos por las organizaciones mundiales de la salud (AU)

Comparación de los efectos provocados por el calor y la irradiación sobre Aspergillus parasiticus / Comparison of effects resulting from the application of physical treatments to Aspergillus parasiticus

El propósito de este trabajo fue estudiar la inhibición de la produción de aflatoxinas asociadas al crecimiento del hongo Aspergillus parasiticus NRRL 2999, mediante el empleo de radiaciones ionizantes. El hongo fue cultivado en arroz de tipo comercial en condiciones de temperatura y humedad ideales para la producción de toxinas. Los cultivos así obtenidos fueron irradiados con dosis de 1,5 kGy* (2D10) al cabo de distintos tiempos de desarrollo, observándose que la máxima inhibición de la producción de toxinas se alcanzaba al irradiar cultivos de 20 horas de desarrollo. Asimismo, se estudió el efecto del calor y su combinación con la irradiación sobre la producción de aflatoxinas, la cual fue seguida a lo largo de 11 días de desarrollo de los cultivos. El contenido de toxinas de los distintos cultivos se dosó por dilución a estinción en cromatografía de capa delgada. Los resultados obtenidos mostraron que, tanto un calentamiento de 15 min a 55-C, como la irradiación de los cultivos con una dosis de 1,5kGy, disminuyen el contenido de toxinas, siendo este último processo el más efectivo. Más aún, la combinación de estos dos agentes físicos, irradiando los cultivos inmediatamente después de ser sometidos al calentamiento, reduce los niveles de toxinas por debajo de los límites de detección sugeridos por las organizaciones mundiales de la salud

DNA strand scission and apurinic sites induced by photoactivated aflatoxins.

Covalent binding of photoactivated aflatoxins to DNA in vitro under close-to-physiological conditions resulted in the formation of apurinic sites and in strand scission. Linearized pBR322 DNA was randomly fragmented, and supercoiled DNA was relaxed during the binding reaction. A primary amine facilitated fragmentation and relaxation. Quantitative measurements of relaxation revealed that the probability of a binding event to be converted into a DNA chain break was approximately 3-5% in the absence of a primary amine. The presence of the latter increased the probability approximately 2- to 3-fold. The results are compatible with the model that photoactivated aflatoxins bind to guanines on DNA; some of these guanine adducts are released, creating apurinic sites. The latter are converted into DNa chain breaks at physiological pH and temperature. Thus, apurinic sites and DNA chain breaks must be considered as quantitatively important (genotoxic) DNA damage induced by aflatoxins.

Effects of gamma irradiation on aflatoxins.

The effects of gamma irradiation on a mixture of aflatoxins B1, B2, G1, G2 were studied. Standard solutions of A and B were irradiated at 5, 10, and 20 kGy in a solution of water/DMSO (9 + 1, v/v) by using a 137Cs source. The control (0 kGy) and irradiated samples were subjected to RP-HPLC analyses with methanol/water (4 + 5, v/v) as the mobile phase. Aflatoxin B1 (AFB1) was the most radio-sensitive of the four compounds. The radiosensitivity of the other aflatoxins, was in increasing order: G2, B2, G1. Only about 5% of AFB1 remained after irradiation of solution A at 5 kGy. When the concentration of solution B was increased two-fold, trace amounts of AFB1 remained after irradiation doses of 10 and 20 kGy. Irradiation was found to be suitable for the destruction of aflatoxins in solution.

Use of ultraviolet energy to degrade aflatoxin M1 in raw or heated milk with and without added peroxide.

Raw whole milk was artificially contaminated to contain 1 ppb aflatoxin M1. A thin layer of milk (.1 cm) was irradiated with ultraviolet energy. In the first experiment, milk was held at 90 degrees C for 10 min, cooled to 20 degrees C, and irradiated for 30 min. Amount of aflatoxin M1 decreased equally (56.2 vs. 53.9%) in raw or preheated milk, suggesting no involvement of milk enzymes in degrading aflatoxin M1 by ultraviolet energy. Data obtained when raw milk containing aflatoxin M1 was exposed to ultraviolet energy for 15 to 60 min suggest first order kinetics for the degradation reaction. In another experiment, milk was held at 5, 25, or 65 degrees C while it was being irradiated. Aflatoxin M1 was degraded at all temperatures. Amount of toxin decreased nonlinearly when temperature at which milk was held was increased. Presence in milk of benzoyl peroxide at .002% did not change the extent to which aflatoxin M1 was degraded by irradiation. Amount of toxin, however, decreased by 89.1% in milk containing .05% H2O2 as compared with 60.7% for H2O2-free milk when both were exposed to ultraviolet irradiation for 20 min at 25 degrees C.

[Microsomal and photochemical oxidation of aflatoxin B1]. / Mikrosomal'noe i fotokhimicheskoe okislenie aflatoksina B1.

The UV irradiation (lambda = 362 nm) of aflatoxin B1 (AfB1) dissolved in water resulted in the formation of an oxidized product. The process was not inhibited by ionol, a routine inhibitor of the radical processes. The oxidized product was not found in the system where AfB1 was metabolized by the 3-methylcholanthrene-activated rat liver microsomes. It is suggested that the product is identical with 2,3-dihydrodiol of AfB1.

Sensitivity of aflatoxin b1 to ionizing radiation.

Aflatoxin B1 in a 5-micrograms/ml water solution was sensitive to ionizing radiation. Inactivation was assayed by the Ames microsome mutagenicity test and confirmed by thin-layer chromatography. Destruction of aflatoxin B1 had already begun at 2.5 kilograms (kGy; 1 kGy = 0.1 Mrad), but a dose exceeding 10 kGy was necessary for total destruction.

Use of sunlight to partially detoxify groundnut (peanut) cake flour and casein contaminated with aflatoxin B1.

Sunlight destroyed 83 and 50% of the toxin added to casein and groundnut cake flour, respectively. Equilibrium dialysis revealed that both casein and groundnut protein bind aflatoxin but the toxin bound to casein appeared more photo-labile than that bound to groundnut protein.