The AFLATOX® Project: Approaching the Development of New Generation,Natural-Based Compounds for the Containment of the Mycotoxigenic Phytopathogen Aspergillus flavus and Aflatoxin Contamination

The control of the fungal contamination on crops is considered a priority by the sanitary authorities of an increasing number of countries, and this is also due to the fact that the geographic areas interested in mycotoxin outbreaks are widening. Among the different pre- and post-harvest strategies that may be applied to prevent fungal and/or aflatoxin contamination, fungicides still play a prominent role; however, despite of countless efforts, to date the problem of food and feed contamination remains unsolved, since the essential factors that affect aflatoxins production are various and hardly to handle as a whole. In this scenario, the exploitation of bioactive natural sources to obtain new agents presenting novel mechanisms of action may represent a successful strategy to minimize, at the same time, aflatoxin contamination and the use of toxic pesticides. The Aflatox^® Project was aimed at the development of new-generation inhibitors of aflatoxigenic Aspergillus spp. proliferation and toxin production, through the modification of naturally occurring molecules: a panel of 177 compounds, belonging to the thiosemicarbazones class, have been synthesized and screened for their antifungal and anti-aflatoxigenic potential. The most effective compounds, selected as the best candidates as aflatoxin containment agents, were also evaluated in terms of cytotoxicity, genotoxicity and epi-genotoxicity to exclude potential harmful effect on the human health, the plants on which fungi grow and the whole ecosystem.     

高效液相色谱-质谱联用法测定薏苡仁药材中黄曲霉毒素

目的建立高效液相色谱-质谱联用法(high performance liquid chromatography-masss pectrometer,HPLC-MS)测定薏苡仁中药饮片中黄曲霉毒素B_1、B_2、G_1、G_2的方法,并对比不同产地薏苡仁中黄曲霉毒素含量。方法样品经过免疫亲合柱处理后,采用HPLC-MS进行测定。分析柱为C18柱(50 mm×2.1 mm, 1.8μm),流动相为10 mmol/L醋酸铵溶液-甲醇溶液,梯度洗脱,流速为0.3 mL/min,柱温为25℃。结果黄曲霉毒素B_1在0.26～10.4ng/mL浓度范围内与峰面积值均呈现良好的线性关系(r~2=0.9995),平均加样回收率为75.4%～123.9%,黄曲霉毒素B2在0.0875～3.5 ng/mL浓度范围内与峰面积值均呈现良好的线性关系(r~2=0.9996),平均加样回收率为71.1%～124.2%,黄曲霉毒素G_1在0.295～11.89 ng/mL浓度范围内与峰面积值均呈现良好的线性关系(r~2=0.9995),平均加样回收率为78.9%～112.2%,黄曲霉毒素G2在0.1475～5.9 ng/mL浓度范围内与峰面积值均呈现良好的线性关系(r~2=0.9992),平均加样回收率为73.8%～123.9%。结论该方法准确、可靠、专属性强,通过精确化合物离子监测,可准确的测定薏苡仁中黄曲霉毒素含量。     

Aflatoxin contamination and exposure in processed cereal-based complementary foods for infants and young children in greater Accra,Ghana

In the study, aflatoxin levels were assessed in thirty five (35) cereal-based food products intended for infants and young children. Additionally, the results showed that 71% of the processed foods intended for infants contained AFB₁ (0.18 ± 0.01 to 36.10 ± 0.32 μgkg⁻¹) levels higher than the European Union permissible limits of 0.1 μg kg⁻¹. Aflatoxin intake was estimated using aflatoxin levels in the food products and the estimated individual consumption rates. The study also revealed mixed cereals as having the highest intake of aflatoxin B₁ contaminants (0.005–0.852 μgkg⁻¹bw d⁻¹; 0.004–0.657 μgkg⁻¹bwd⁻¹) with mean estimated daily intake (EDI) of 0.23 ± 0.16 μgkg⁻¹bwd⁻¹ and 0.153 ± 0.13 μgkg⁻¹bwd⁻¹ for infants and young children respectively. The estimated AFT intake recorded for infants and young children for all the cereal-based food ranged from 0.005 to 1.054 μgkg⁻¹bwd⁻¹ and 0.004–0.838 μgkg⁻¹bwd⁻¹ respectively.     

Effect of contamination of foods by Aspergillus flavus on the nutritive value of protein

The effect of aflatoxin was measured on the protein quality of peanut meal (PNM) and fish meal (FM) Total protein efficiency, protein efficiency ratio, net protein utilisation, examination of the histopathology of the liver, ileal digesion of amino acids and plasma amino acid concentration were used as bioassays together with chemical score (CS), dye binding capacity (DBC), essential amino acid index (EAAI), and discriminant computered PER (DC-PER) as chemical methods. In trial 1, aflatoxin-free PNM was compared with infected PNM at graded levels of toxin when fed to chickens and ducklings. In trial 2, various mixtures of PNM and FM at a constant aflatoxin level (280 μg kg^?1) were fed to compare the effects of aflatoxin on proteins of differing quality. Ducks were more sensitive to the toxin than chickens, as indicated by deterioration of protein quality, and the effects on growth and the histological appearance of the liver were magnified on diets of low quality (PNM), but not of high quality (FM). Contamination of PNM resulted in progressive increase in DBC and, to a lesser extent, in DC-PER, while EAAI and CS were not affected. The importance of these findings lies in the problems of mould contamination of animal feedstuffs in humid, tropical conditions, which may affect the more sensitive animals, and may not be detected by chemical methods of measuring protein quality, nor by bioassay on chickens, if the levels of contamination are low.     

Survey of aflatoxins in rice from Iran using immunoaffinity column clean-up and HPLC with fluorescence detection

A study was undertaken to determine levels of aflatoxins in rice. A total of 261 rice samples were analyzed by HPLC using a method was based on the extraction of 50?g of finely ground rice plus 5?g?NaCl with 200?ml of 80% methanol. After filtration and immunoaffinity clean-up, 20?µl was injected onto the HPLC. HPLC analysis was carried out using a Genesis RP C₁₈ column (250?×?4.6, 4?µm I.D.) and a mobile phase with a linear gradient of water/methanol/acetonitrile (6?:?2?:?2?v/v) over 16?min. Aflatoxins were determined after post-column derivatisation with iodine by fluorescence detection at excitation and emission wavelengths of 365 and 445?nm, respectively. It was found that 68.9% of the rice samples contained aflatoxin B₁ at levels greater than 0.2?ng?g^?1.     

Contamination by moulds of grape berries in Slovakia

This paper describes the first map, albeit partial, of toxigenic fungi re-isolated from grape berries collected in three out of the six most important Slovakia winemaking areas in two different periods of the harvest year 2008. Low temperatures and high relative humidity during July 2008 favoured the development of grape fungal diseases that cause rots such as Plasmopara, Uncinula, Botrytis, Metasphaeria, Elsinoë, and Saccharomycetes. In the analysed samples, the following genera of toxigenic fungi were identified in the range of 1–4%: Aspergillus, Alternaria, Cladosporium, Epicoccum, Fusarium, Penicillium, Rhizopus, Ulocladium, and Trichoderma Trichothecium, while the genera Aspergillus, Alternaria, Fusarium, and Penicillium were in the range 11–29%. A. niger, A. carbonarius, some strains of A. carbonarius–with ‘crystals’ and strains of A. uvarum–uniseriate were identified; these species are considered ochratoxigenic (able to produce variable amounts of toxins). In addition, a non-ochratoxigenic strain of A. ibericus and a Fusarium strain able to biosynthesize small amount of fumonisins, beauvericin, and enniatins were identified. P. expansum, able to produce citrinin, represents 29.7%, of the Penicillium genus together with P. verrucosum, P. glabrum, P. citrinum, and P. crustosum. An analysis for the identification and quantification of the main toxins: ochratoxin A, fumonisins, beauvericin, enniatins, and fusaproliferin was performed on grape samples; it was consistent with the results of the mycological analysis. Toxigenic fungi should be checked throughout the years and their occurrence compared with all environmental factors to avoid health risks.     

Aflatoxins and ochratoxin A in tea prepared from naturally contaminated powdered ginger

The migration of several major mycotoxins, aflatoxins B₁ (AFB₁), B₂, G₁, and G₂ (AFT, total of the aflatoxins) and ochratoxin A (OTA), from naturally contaminated powdered ginger to surrounding liquid (tea) was investigated. The toxins are commonly found in cereal grains and are toxic, carcinogenic and thermostable. Ginger root is widely used for digestive problems. Powdered ginger (2 g) found to contain AFT and OTA was placed in an empty heat sealable tea bag. The tea bag was heat-sealed and used to prepare tea under different conditions: temperature (50 and 100°C), time (5 and 10 min) and volume (100 and 200 ml). The tea bag was placed in hot water and stirred every 1 min for 5 s during the first 5 min of steeping. After steeping, the tea bag was removed and the tea and ginger residue (in the tea bag) were analysed separately for AFT and OTA. After extraction and immunoaffinity column (IAC) clean-up, the isolated AFT and OTA were separated by reversed-phase liquid chromatography and quantified using a fluorescence detector. At 100°C, approximately 30–40% of AFB₁ and AFT and 20–30% of OTA in the contaminated ginger were found in the ginger tea; the total amounts of AFT and OTA in tea varied less than 5% under the three conditions of preparation. At 50°C, about 10% of OTA and AFT were found in tea. This is the first study on the migration of AFT from botanicals to tea. It is also the first to study the distribution of AFT and OTA from powdered ginger to tea and ginger residue.     

Indirect determination of aflatoxin B1 in beer via a multi-commuted optical sensor

This paper reports the determination of aflatoxin B₁ (AFB₁), one of the most carcinogenic substances known. A multi-commuted flow injection–solid phase spectroscopy (FI–SPS) system combined with photochemically induced fluorescence (PIF) was developed, for the first time, for its quantitative determination. A strongly fluorescent degradation product was obtained on-line by irradiation with ultraviolet light. The determination was carried out by measuring the fluorescence intensity of the photo-product at 353/424 (λ _ex/λ _em), once retained on C₁₈ silica-gel filling the flow-cell. A linear dynamic range of 0.09–12?µg?l^?1, detection limit as sensitive as 29?ng?l^?1 and a relative standard deviation (RSD) of 1.4% were obtained. The method proposed was satisfactorily applied to the determination of AFB₁ in different types of beer (normal and non-alcoholic). Hydrophobic compounds were eliminated from beer samples and AFB₁ was extracted with acetonitrile by solid-phase extraction on C₁₈ sorbent. Recoveries of the target compound from spiked beers were between 94 and 106%. The results obtained in the analysis of real samples are in good agreement with those provided by a reference chromatographic method.     

Analysis of industry-generated data. Part 2: Risk-based sampling plan for efficient self-control of aflatoxin M1 contamination in raw milk

Aflatoxin M₁ (AFM1) contamination in 21 969 milk samples taken in Italy during 2005–08 and 2010 provided the basis for designing an early warning self-control plan. Additionally, 4148 AFM1 data points from the mycotoxin crisis (2003–04) represented the worst case. No parametric function provided a good fit for the skewed and scattered AFM1 concentrations. The acceptable reference values, reflecting the combined uncertainty of AFM1 measured in consignments consisting of milk from one to six farms, ranged from 40 to 16.7 ng kg^?1, respectively. Asymmetric control charts with these reference values, 40 and 50 ng kg^?1 warning and action limits are recommended to assess immediately the distribution of AFM1 concentration in incoming consignments. The moving window method, presented as a worked example including 5 days with five samples/day, enabled verification of compliance of production with the legal limit in 98% of the consignments at a 94% probability level. The sampling plan developed assumes consecutive analyses of samples taken from individual farms, which makes early detection of contamination possible and also immediate corrective actions if the AFM1 concentration in a consignment exceeds the reference value. In the latter case different control plans with increased sampling frequency should be applied depending on the level and frequency of contamination. As aflatoxin B₁ increases in feed at about the same time, therefore a coordinated sampling programme performed by the milk processing plants operating in a confined geographic area is more effective and economical then the individual ones. The applicability of the sample size calculation based on binomial theorem and the fast response rate resulting from the recommended sampling plan were verified by taking 1000–10 000 random samples with replacement from the experimental databases representing the normal, moderately and highly contaminated periods. The efficiency of the control plan could be substantially enhanced if the dairy farms used feed with a tolerable level of AFB1.