Determination of fumonisin B1 in bovine milk by LC–MS/MS

The aim of the present work was to develop a sensitive and selective method for identification and quantification of fumonisin B1 (FB1) in bovine milk. FB1 was isolated by immunoaffinity column and was detected using liquid chromatography coupled with tandem mass spectrometry in positive electrospray ionization (ESI+).The LOQ of the method was 0.1 μg/kg that was lower than the others reported in the literature. The high coefficient of determination (R² > 0.99) obtained in the range of 0.1–10.0 μg/kg, the good recovery (84%) and relative standard deviation (7%) of the proposed method ensure correct fumonisin detection in milk even at relatively low concentrations.The developed method was applied on different commercial samples in order to test its efficacy. FB1 was found above the LOQ in eight out of 10 samples analysed and the average level of contamination found was 0.26 μg/kg.     

Curcuma longa L. essential oil composition,antioxidant effect,and effect on Fusarium verticillioides and fumonisin production

Essential oils may be an alternative to the use of synthetic fungicides for the control of fungi involved in agricultural product deterioration. The aim of the present study was to evaluate the composition and antioxidant effect of turmeric essential oil and its antifungal and antimycotoxigenic action on Fusarium verticillioides (Sacc.) Nirenberg. The essential oil major components were α-turmerone (42.6%), β-turmerone (16.0%) and ar-turmerone (12.9%). The half-maximal inhibitory concentration (IC₅₀) for the radical scavenging capacities of 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were 0.54 and 10.03 mg/ml, respectively, indicating good antioxidant activity. The application of 17.9 and 294.9 μg/ml of turmeric essential oil decreased the development of F. verticillioides by 56.0 and 79.3%, respectively, when compared with the fungal control. The scanning electron microscopy demonstrated that the oil decreased the thickness and the length of the microconidia. Ergosterol production significantly decreased (p < 0.05) in groups treated with the essential oil relative to the control, indicating an effect of the oil on fungal biomass. The production of B₁ and B₂ fumonisins was significantly inhibited (p < 0.05) in groups treated with the essential oil. The results suggest that turmeric essential oil has antioxidant, antifungal and antimycotoxigenic activities.     

Further data on the occurrence of Fusarium emerging mycotoxins enniatins (A,A1, B,B1), fusaproliferin and beauvericin in raw cereals commercialized in Morocco

In this study, 64 samples of raw cereals (wheat, maize and barley) purchased from local markets in Rabat–Salé area from Morocco were analyzed for the occurrence of six emerging mycotoxins: four enniatins ENs (ENA, ENA1, ENB and ENB1), beauvericin (BEA) and fusaproliferin (FUS). Samples were extracted with a mixture of water/acetonitrile (85/15, v/v) by using an Ultra-turrax homogenizer. Mycotoxins were then identified and quantified with a liquid chromatography (LC) with diode array detector (DAD). Positive samples were confirmed with an LC–MS/MS. Analytical results showed that the frequencies of contamination of total samples with ENs, BEA and FUS were 50, 26.5 and 7.8%, respectively. ENA1 was the most common EN found with a percentage of contamination of 39%, levels ranged between 14 and 445 mg/kg. ENB contaminated 14 samples (21.8%) and levels ranged from 5 to 100 mg/kg. ENB1 was present in four samples (6.2%) and levels varied from 8 to 32 mg/kg. ENA was detected in only one sample with 34 mg/kg. BEA levels ranged from 1 to 59 mg/kg and FUS levels varied from 0.6 to 2 mg/kg. The present report is the first one ever drafted on the presence of emerging Fusarium mycotoxins in raw cereals available in Morocco.     

Development and validation of an accurate and rapid LC-ESI-MS/MS method for the simultaneous quantification of aflatoxin B1, B2, G1 and G2 in lotus seeds

An accurate and rapid LC-ESI-MS/MS analytical method was developed and validated for the simultaneous determination of aflatoxin B₁, B₂, G₁ and G₂ in lotus seeds. The samples were firstly extracted with methanol-water solution (80:20, v/v), and then cleaned up by immunoaffinity columns. The mass spectrometer was operated in the positive ionization electrospray (ESI+) mode using multiple reaction monitoring (MRM) for analysis of four aflatoxins. The transitions of m/z 313 → 285 (AFB₁, CE 33 eV), m/z 315 → 259 (AFB₂, CE 37 eV), m/z 329 → 243 (AFG₁, CE 37 eV) and m/z 331 → 257 (AFG₂, CE 37 eV) were used to quantify these four aflatoxins, respectively. The limits of detection (LODs) of aflatoxin B₁, B₂, G₁ and G₂ were 0.007, 0.005, 0.003 and 0.005 μg kg^?1 based on a signal-to-noise ratio of 3:1, respectively. The limits of quantification (LOQs) of aflatoxin B₁, B₂, G₁ and G₂ were 0.02, 0.015, 0.01 and 0.015 μg kg^?1 based on a signal-to-noise ratio of 10:1, respectively. Recoveries for samples of spiked lotus seeds were all above 66% with relative standard deviation all below 15% for all compounds. Nineteen out of twenty batches of lotus seeds collected from different drug stores or markets in China were found to be contaminated with aflatoxins at different levels ranging from 0.02 to 688.4 μg kg^?1.     

First report on the presence of emerging Fusarium mycotoxins enniatins (A,A1, B,B1), beauvericin and fusaproliferin in rice on the Moroccan retail markets

Seventy samples of rice purchased from local markets in six cities from Morocco (Rabat, Casablanca, Kénitra, Mohammadia, Tanger and Errachidia) were analyzed for the presence of six emerging mycotoxins: four enniatins ENs (ENA, ENA₁, ENB and ENB₁), beauvericin (BEA) and fusaproliferin (FUS). Samples were extracted with a mixture of acetonitrile/water (85/15, v/v) by using an ultra-turrax homogenizer. Mycotoxins were then identified and quantified with liquid chromatography (LC) coupled to diode array detector (DAD). Positive samples were confirmed with an LC-MS/MS. Analytical results showed that BEA was present in 75.7% of total analyzed samples. BEA levels varied between 3.8 and 26.3 mg/kg. The frequencies of contamination of samples with total ENs and FUS were 50% and 4.3%, respectively. Among the ENs, ENB was the mycotoxin much more found (30% of total samples), while ENB₁, ENA and ENA₁ were found in 24.6%, 22.8% and 5.7% of total samples, respectively. The high ENs value was registered in a rice sample from kénitra (448.7 mg/kg of ENA₁). This is the first study that describes the presence of emerging Fusarium mycotoxins in rice available in Morocco.     

Biocontrol of dairy moulds by antagonistic dairy yeast Debaryomyces hansenii in yoghurt and cheese at elevated temperatures

Some strains of the yeast Debaryomyces hansenii are known to be antagonistic toward moulds. In this study, we describe the inhibitory effects of a dairy strain of D. hansenii as a biocontrol agent against a number of dairy moulds in plain yoghurt and cheese under non-refrigerated conditions. This antagonistic yeast showed inhibition of growth of the following dairy moulds: Aspergillus sp., Byssochlamys fulva, Byssochlamys nivea, Cladosporium sp., Eurotium chevalieri, Penicillium candidum and Penicillium roqueforti. However, the inhibitory effect of this antagonistic yeast against dairy moulds is dependent upon the concentration of the moulds: the lower the concentration, the more effective the yeast. The findings of this study have implications for both cheese maturation and dairy biopreservation. Good manufacturing practice and hygiene to keep the contaminant load down is essential for the prevention of dairy spoilage.     

Multilayer perceptron neural networks and radial-basis function networks as tools to forecast accumulation of deoxynivalenol in barley seeds contaminated with Fusarium culmorum

The capacity of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict deoxynivalenol (DON) accumulation in barley seeds contaminated with Fusarium culmorum under different conditions has been assessed. Temperature (20–28 °C), water activity (0.94–0.98), inoculum size (7–15 mm diameter), and time were the inputs while DON concentration was the output. The dataset was used to train, validate and test many ANNs. Minimizing the mean-square error (MSE) was used to choose the optimal network. Single-layer perceptrons with low number of hidden nodes proved better than double-layer perceptrons, but the performance depended on the training algorithm. The RBFN reached lower errors and better generalization than MLP-ANN but they required a high number of hidden nodes. Accurate prediction of DON accumulation in barley seeds by F. culmorum was possible using MLP-ANNs or RBFNs.     

Aflatoxin and fumonisin contamination of yam flour from markets in Nigeria

The natural occurrence of aflatoxins (AFs) and fumonisins (FBs) in yam flour samples (n = 100) obtained in south-western Nigeria was evaluated. AFs were determined by HPLC with fluorescence detection and FBs by HPLC coupled with mass spectrometry. Aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁) were found in 57% and 21% of flours from white yam with concentrations ranging from <0.02 (limit of detection, LOD) to 3.2 μg kg⁻¹ (mean = 0.4 μg kg⁻¹) and from <0.05 to 3.5 μg kg⁻¹, respectively. AFB₁ was the only aflatoxin detected in samples from water yam, contaminating 32% of the samples with values ranging from <LOD to 0.6 μg kg⁻¹ (mean = 0.1 μg kg⁻¹). Fumonisin B₁ was found in 32% of the white yam samples (<0.5 (LOD) to 91 μg kg⁻¹; mean = 5 μg kg⁻¹) and in 5% of water yam samples (<LOD to 2 μg kg⁻¹). AFs and FBs were significantly higher (P < 0.05) in white yam flours compared to water yam flours. Preparation of amala from naturally-contaminated yam flour resulted in reduction of AFB₁ and AFG₁ by 44% and 51% respectively. From this study, only 7% of the samples contained AFs above the European standard limits for cereals intended for direct human consumption, while all the FBs-positive samples were well below the limits. The occurrence of ochratoxin A, zearalenone and deoxynivalenol was also evaluated in 20 samples; these mycotoxins were never detected.     

Biological detoxification of Aflatoxin B1 by Bacillus licheniformis CFR1

Aflatoxin B₁ is the most harmful among the mycotoxins commonly present in food and feed, and it may lead to hepatocellular carcinoma in humans and animals. Therefore, limiting its exposure to humans and livestock is very much essential. The present study aims to isolate and characterize Aflatoxin B₁ detoxifying bacteria from various sources, to develop a safe and environment-friendly strategy for Aflatoxin B₁ management. Fifty-six bacteria were isolated using a media amended with coumarin as a sole carbon source. Seven strains showed more than 70% reduction of AFB₁ in liquid culture media. Among them, isolate CFR1 reduced Aflatoxin B₁ by 94.7%, and it was selected for further studies. CFR1 was identified as Bacillus licheniformis CFR1, by biochemical characterization and 16S rRNA gene sequencing. The cell-free supernatant of B. licheniformis CFR1 was able to degrade AFB₁ efficiently than the cell lysate. The degradation of AFB₁ was examined using High-Performance Thin Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Electron spray ionization-Mass Spectrometry (ESI-MS). The optimal temperature, time, and pH of the medium for the maximum degradation of Aflatoxin B₁ were found to be 37 °C, 24 h and 7, respectively. Furthermore, Ames test for mutagenicity showed that when treated with B. licheniformis CFR1 extracellular fraction coincided with the loss of Aflatoxin B₁ mutagenicity. To the best of our knowledge, this is the first study that shows more than 90% degradation of AFB₁ by B. licheniformis. Thus, B. licheniformis CFR1 might be an excellent candidate for bioremediation and detoxification of Aflatoxin B₁ from both field and food matrices.     

埕北油田B平台锅炉控制系统PLC改造

PLC是一种基于工控机的自动控制装置,所有的内部逻辑通过其内部软件实现,以提高控制系统的可靠性和可诊断性.埕北油田B平台锅炉控制系统改造为PLC控制后,原系统存在的无法正常启动、无故障自动停机、程控器走时不准和停机后无法热启动等问题得到了彻底解决.     

埕北油田B平台锅炉控制系统PLC改造

PLC是一种基于工控机的自动控制装置,所有的内部逻辑通过其内部软件实现,以提高控制系统的可靠性和可诊断性。埕北油田B平台锅炉控制系统改造为PLC控制后,原系统存在的无法正常启动、无故障自动停机、程控器走时不准和停机后无法热启动等问题得到了彻底解决。     

Development of a PCR protocol to detect patulin producing moulds in food products

Patulin is a secondary toxic metabolite with important health effects. Several mould species of Penicillium and Aspergillus genera associated with patulin production have been detected in food products. Thus, specific and sensitive methods to detect patulin producing moulds are needed. The aim of this work was to develop a polymerase chain reaction (PCR) method to detect patulin producing moulds in food. 34 patulin producing and 30 non-producing strains belonging to the main species usually reported in food products were used. Patulin production was firstly evaluated by mycellar electrokinetic capillary electrophoresis and high-pressure liquid chromatography-mass spectrometry in all tested strains. Biosynthesis was also used to develop PCR primers derived from the genes involved in patulin. By means of a primer pair based on the isoepoxydon dehydrogenase (idh) gene, a 496-bp amplicon was specifically detected in all the mould strains previously confirmed as patulin producing, regardless of their genus and species. With the developed method it was possible to detect down to 0.5 ng of pure DNA from producing strains and from 1.8 × 10² to 2.7 × 10³ conidia g^?1 in artificially inoculated foods. No relevant PCR inhibition due to food matrices was observed. The PCR protocol developed could be considered as an appropriate tool to detect patulin producing moulds in food products.     

Impact of Pediococcus pentosaceus strain L006 and its metabolites on fumonisin biosynthesis by Fusarium verticillioides

The aim of this work was to study the effectiveness of a Pediococcus pentosaceus strain L006, isolated from maize leaf and previously characterised for its high antifungal efficiency, on fumonisin biosynthesis by Fusarium verticillioides. Studies performed in GYEP medium supplemented with amylopectin showed a significant increase in fumonisin production when the F. verticillioides strain was simultaneously co-inoculated with the P. pentosaceus strain or inoculated in a three-day-old culture of this lactic acid bacteria. Our studies also demonstrated that some extracellular metabolites produced in MRS medium by the P. pentosaceus strain L006 were able to significantly reduce fumonisin production in liquid medium as well as on maize kernels. Fumonisin yields by F. verticillioides inoculated on autoclaved maize kernels were reduced by a factor ranging from 75% to 80% after 20 days of incubation. Our results illustrate the potential risk linked to the use of an antagonistic bacterial agent to manage fumonisin contamination, while emphasizing the potential use of bacterial metabolites to counteract fumonisin accumulation in kernels.     

Development of a PCR protocol to detect ochratoxin A producing moulds in food products

Ochratoxin A (OTA) is a mycotoxin produced by several Penicillium and Aspergillus species growing in food commodities. To prevent OTA in foods it is necessary to have rapid and specific methods for early detection of producing moulds regardless of species and genera. In this work a PCR method to detect ochratoxigenic moulds has been developed. For this purpose, 75 mould strains belonging to species usually reported in food products were used. Their OTA production was checked by micellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A specific amplicon of 459 bp was detected by using the designed PCR protocol only in the OTA producing strains. The detection limit of the developed PCR protocol was estimated for 25 pg of mould DNA from pure cultures and from about 10²–10⁴ cfu/g when it was evaluated directly on artificially inoculated food. Its functionality in naturally infected samples was also demonstrated. In conclusion, the developed PCR method could be used for detecting ochratoxigenic moulds in foods and consequently for monitoring these moulds in the HACCP programs.     

Optimising sorting and washing of home-grown maize to reduce fumonisin contamination under laboratory-controlled conditions

Subsistence farming communities with low socio-economic status reliant on a mono cereal maize diet are exposed to fumonisin levels that exceed the provisional maximum tolerable daily intake of 2 μg kg^?1 body weight day^?1 recommended by the Joint FAO/WHO Expert Committee on Food Additives. In the rural Centane magisterial district, Eastern Cape Province, South Africa, it is customary during food preparation to sort visibly infected maize kernels from good maize kernels and to wash the good kernels prior to cooking. However, this customary practice seems not to sufficiently reduce the fumonisin levels. This is the first study to optimise the reduction of fumonisin mycotoxins in home-grown maize based on customary methods of a rural population under laboratory-controlled conditions. Maize obtained from subsistence farmers was analysed for the major naturally occurring fumonisins (FB₁, FB₂ and FB₃) by fluorescence HPLC. Large variations were observed in the unsorted and the experimental maize batches attributable to the non-homogeneous distribution of fumonisin contamination in maize kernels. Optimised hand-sorting of maize kernels by removing the visibly infected/damaged kernels (fumonisins, 53.7 ± 15.0 mg kg^?1, 2.5% by weight) reduced the mean fumonisins from 2.32 ± 1.16 mg kg^?1 to 0.68 ± 0.42 mg kg^?1. Hand washing of the sorted good maize kernels for a period of 10 min at 25 °C resulted in optimal reduction with no additional improvement for wash periods up to 15 h. The laboratory optimised sorting reduced the fumonisins by 71 ± 18% and an additional 13 ± 12% with the 10 min wash. Based on these results and on local practices and practicalities the protocol that would be recommended to subsistence farmers consists of the removal of the infected/damaged kernels from the maize followed by a 10 min ambient temperature water wash.     

Survey of aflatoxin B1 in helva,a traditional Turkish food,by TLC

A total of 102 helva samples consisting of 34 plain helva, 34 helva containing cacao, and 34 helva containing pistachio nuts purchased from helva-factories and supermarkets in Adana of Turkey were analysed for aflatoxin B₁ (AFB₁) by thin-layer chromatography. The detection limit of AFB₁ was 1 μg kg⁻¹. Recovery experiments were carried out with spiked samples in the range 2–10 μg kg⁻¹ of AFB₁. No AFB₁ was found in any plain helva and helva containing cacao samples. On the other hand, of 34 helva containing pistachio nuts AFB₁ was determined in eight samples. AFB₁ was found in excess of Turkish legal limit of 5 μg kg⁻¹ in 4 of 102 helva samples. This paper reports the data of the first survey for the presence of AFB₁ in helva in Turkey.     

Aflatoxin B1 degradation by Bacillus subtilis UTBSP1 isolated from pistachio nuts of Iran

Pistachio nuts are among the commodities with the highest risk of aflatoxin contamination in Iran. Aflatoxin B1 (AFB1) is one of the most hazardous mycotoxins for humans and livestock. In nature, there are microorganisms which are capable of reducing aflatoxins contamination in food and feed products. In this study, Bacillus subtilis strain UTBSP1 was isolated from pistachio nuts and studied for the degradation of AFB1. The AFB1 contents were determined by the use of HPTLC and HPLC as well as multiple reactions monitoring (MRM) method in LC-MS/MS. The results indicated B. subtilis UTBSP1 could considerably remediate AFB1 from nutrient broth culture and pistachio nut by 85.66% and 95%, respectively. Cell free supernatant fluid caused an apparent 78.39% decrease in AFB1 content. The optimal conditions for AFB1 degradation by cell free supernatant appeared at 35-40 °C, during 24 h. Furthermore, the results indicated that AFB1 degradation is enzymatic and responsible enzymes are extracellular and constitutively produced. The destructive AFB1 differed from standard AFB1 chemically, and lost a fluorescence property.     

Estimation of dietary intakes of fumonisins B1 and B2 from conventional and organic corn

The dietary intakes of fumonisins from 60 samples of conventional and organic corn were assessed. A 13.3% of the conventional corn samples contained fumonisin B₁ and B₂ at mean levels of 43 and 22 ng/g, respectively, while 10% of the organic corn samples contained fumonisins at somewhat lower levels of 35 ng/g (FB₁) and 19 ng/g (FB₂). Overall, the fumonisin levels in the corn samples were much lower than the maximum level of 2000 ng/g (as the sum of FB₁ and FB₂) proposed for unprocessed maize in a recent EU regulation. The fumonisins present in conventional and organic maize are estimated to contribute with very low percentages of 0.21% and 0.17%, respectively, to the level considered at risk for human health. Based on the data exposed in this paper, the farming system is probably not of decisive importance for the final contamination of agricultural products with these mycotoxins.     

Pressurized fluid extraction for quantitative recovery of aflatoxins B1 and B2 from pistachio

A pressurized fluid extraction method for the extraction of aflatoxins B₁ and B₂ from contaminated pistachio samples was developed using a modified supercritical fluid extractor. The parameters of temperature, pressure, and flow rate of the solvent were optimized for the extraction process. The pressure variation in the range of (10–100) bar had an insignificant effect on the aflatoxins extraction yield. Solution of 80% (v/v) methanol in water as the extraction solvent at flow rate of 0.5 mL/min and temperatures of higher than 80 °C were obtained for efficient extraction of aflatoxins B₁ and B₂ from spiked and naturally contaminated pistachio samples. The developed method in comparison with the AOAC method offered the repeatability (RSD) of 13.5% and 12%, respectively, while the extraction yield or recovery of the analytes was about 20% higher corresponding to those obtained with the AOAC method. The higher recovery of the developed method was validated by HPLC analysis and it was also applied to the peanut samples analysis.