Occurrence of beauvericin and enniatins in wheat flour and corn grits on the Japanese market,and their co-contamination with type B trichothecene mycotoxins

The contamination levels of beauvericin and four enniatins, A, A₁, B and B₁, in 207 samples of wheat flour and corn grits on the Japanese market were determined by an analytical method based on LC-MS/MS. The toxins were extracted from samples with acetonitrile–water (85:15, v/v) and then purified with C18 cartridges. The method was validated in a single laboratory using spiked samples at two levels; the recovery of the five toxins ranged from 91.1% to 113.8%. Enniatin B was frequently detected in imported wheat flour (81.8%) and domestic wheat flour (85.6%), and the highest concentration of enniatin B was present in a domestic wheat sample (633 μg kg^?1). In corn grits, beauvericin was found in 34% of the samples, but enniatins were not detected at all. The maximum concentration of beauvericin in corn grits was 26.1 μg kg^?1. Deoxynivalenol and nivalenol in the same samples were determined by a method using an immunoaffinity column. Co-contamination of deoxynivalenol and enniatins was observed in 61% of the imported wheat samples and in 58% of the domestic wheat samples. These results suggest the need for a risk assessment for cyclic depsipeptide mycotoxins in Japan and a study on the synergistic effect of deoxynivalenol and enniatins.     

食品中白僵菌素和恩镰孢菌素的污染情况及分析方法研究进展

介绍了食品中白僵菌素(beauvericin,BEA)和包括恩镰孢菌素A(enniatin A,ENA)、恩镰孢菌素A_1(enniatin A_1,ENA_1)、恩镰孢菌素B(enniatin B,ENB)、恩镰孢菌素B_1(enniatin B_1,ENB_1)在内的4种主要的恩镰孢菌素(enniatins,ENNs)的分类、毒性和分析方法,尤其是前处理方式和各方法定量限的研究进展。综述了西班牙、摩洛哥、意大利、日本等部分国家食品中BEA和4种ENNs的污染状况以及这5种毒素与其他主要真菌毒素的协同污染情况。提出了建立针对复杂食品基质中BEA和ENNs测定的高效液相色谱-串联质谱法。     

Determination of Fusarium mycotoxins beauvericin and enniatins (A,A1, B,B1) in eggs of laying hens using liquid chromatography–tandem mass spectrometry (LC–MS/MS)

An existing sample preparation technique used for the determination of ionophoric coccidiostats was modified to permit the analysis of Fusarium mycotoxins beauvericin and enniatins in egg samples. The validation results indicated that the sample preparation method developed is well applicable to the determination of the related compounds in eggs. The presence and contamination levels of beauvericin and enniatins A, A1, B and B1 were studied in Finnish egg samples in 2004–2005. The egg sample analyses (112 whole eggs and 367 egg yolk) revealed that the occurrence of beauvericin as well as enniatins B and B1 is very common in Finnish eggs. The contaminations were, however, in most cases in trace-levels (<limit of quantification). Enniatin A and A1 were not found in any of the whole egg samples, and furthermore enniatin B1 was present only in samples from 2004. The general contamination levels of beauvericin and enniatins in whole egg samples were similar in 2004 and 2005. The prevalence and concentration levels of mycotoxins were higher in the market samples (egg yolk) as compared to samples collected in the national residue monitoring programme (whole egg) samples suggesting that there may be bioaccumulation of these mycotoxin contaminants in egg yolk. This is the first study to report the presence of Fusarium mycotoxins beauvericin and enniatins in egg samples.     

Presence and concentrations of the Fusarium-related mycotoxins beauvericin, enniatins and moniliformin in finnish grain samples

Fusarium mycotoxins beauvericin, enniatins (A, A1, B, B1) and moniliformin were analysed in 38 Finnish grain samples (14 wheat, 22 barley, one rye, one oats) harvested in 2001-02. The contaminating Fusarium species were identified with the primer-specific polymerase chain reaction as well as with morphological studies. All the studied mycotoxins were found in the samples. Enniatins B and B1 were detected in all samples, and enniatin A, enniatin A1, beauvericin and moniliformin in 74, 95, 95 and 74% of the samples, respectively. There were higher concentrations of the mycotoxins analysed in 2001 compared with 2002. The highest levels of mycotoxins were detected in samples harvested late in the autumn after a long rainy period. Fusarium avenaceum was the most abundant Fusarium species in Finland during both years (0-29.5%) measured as infected kernels. A significant correlation was found between F. avenaceum contamination level and the concentration levels of enniatins B and B1, as well as moniliformin.     

Multi-mycotoxins Analysis in Dried Fruit by LC/MS/MS and a Modified QuEChERS Procedure

A sensitive and reliable multi-mycotoxin method was developed for the simultaneous determination of 16 toxicological important mycotoxins, such as aflatoxins B₁, B₂, G₁, and G₂; enniatins A, A₁, B, and B₁; beauvericin; ochratoxin A; fumonisin B₁, B₂, and B₃; diacetoxyscirprenol; HT-2; and T-2 toxin in dried fruits using liquid chromatography combined with electrospray ionization-triple quadrupole tandem-mass spectrometry. Mycotoxins have been extracted from the samples using a modified quick, easy, cheap, effective, rugged, and safe procedure. The method was based on a single extraction with acidified acetonitrile, followed by partitioning with salts, avoiding any further clean-up step. Limits of detections ranged from 0.08 to 15 μg kg^?1 and limits of quantification ranged from 0.2 to 45 μg kg^?1, which were below the legal limit set by the European Union for the legislated mycotoxines. The recoveries in spiked samples ranged from 60 to 135 % except for beauvericin using matrix-matched calibration curves for quantification, with good inter- and intraday repeatability (respective relative standard deviation ≤20 and 9 %). The developed method was applied to 15 commercial dried fruits: raisins, figs, apricots, plums, and dates purchased in local markets from Spain. Among the mycotoxins studied, enniantins and aflatoxins were the most predominant mycotoxins.     

Beauvericin and enniatins A,A1, B and B1 in Norwegian grain: a survey

Norwegian grain samples (73 oats, 75 barley, 80 wheat) from the 2000 to 2002 growing seasons were examined for contamination with five different enniatins and the association between the found concentrations and the prevalence or infection level with several common Fusarium species investigated. Enniatin B was the fungal metabolite with the highest prevalence (100%) and the highest maximum concentration (5800 μg/kg, wheat). The maximum concentration of all five enniatins together in a single sample was 7400 μg/kg (wheat). Enniatin concentrations were correlated with several independent variables, among them grain species. Beauvericin was only sporadically detected in barley and wheat and at concentrations just above the limit of detection of 3 μg/kg, while amounts up to 120 μg/kg were found in oats. The likelihood of detecting enniatin A1 as well as the concentrations of enniatins B and B1 could be mainly related to infection with Fusarium avenaceum/arthrosporioides, and the likelihood of detecting beauvericin could be related to infection with Fusarium poae. This survey indicates that the prevalence of enniatins A1, B and B1 in Norwegian grain is high, and that enniatin B concentrations of above 1000 μg/kg are common in barley and wheat.     

Further data on the levels of emerging Fusarium mycotoxins enniatins (A,A1, B,B1), beauvericin and fusaproliferin in breakfast and infant cereals from Morocco

Sixty-eight samples of cereals products, including breakfast cereals (n = 48) and infant cereals (n = 20), purchased from supermarkets and pharmacies in Rabat-Salé area from Morocco were analysed for the determination 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), using an Ultra-Turrax® homogeniser. Mycotoxins were then identified and quantified by liquid chromatography (LC) with diode array detection (DAD). Positive samples were confirmed by LC–MS/MS.     

Development of a new method for the simultaneous determination of 21 mycotoxins in coffee beverages by liquid chromatography tandem mass spectrometry

A new method for the simultaneous detection of 21 mycotoxins (ochratoxin A, aflatoxin B₁, aflatoxin B_2, aflatoxin G₁, aflatoxin G₂, sterigmatocystin, nivalenol, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, diacetoxyscirpenol, neosolaniol, HT-2 toxin, T-2 toxin, fumonisin B₁, fumonisin B₂, enniatin A, enniatin A₁, enniatin B, enniatin B₁, and beauvericin) in coffee beverages was internally validated. The method is based on liquid/liquid extraction with a mixture of ethyl acetate/formic acid (95:5 v/v) and detection using triple quadrupole (QqQ) and ion trap (IT) liquid chromatography tandem mass spectrometry. The limits of detection and quantification were 0.02 to 39.64 μg/kg, respectively, and the correlation coefficients were optimal for all mycotoxins (R² ≥ 0.992). The recovery values ranged from 72% to 97%. The developed method was demonstrated in six real samples of roasted and instant coffee, caffeinated and decaffeinated coffee, and coffee with sugar added. The analyses indicate the presence of the studied mycotoxins in coffee beverages at μg/kg concentrations. Ochratoxin A, a mycotoxin that is regulated in coffee, was detected in two samples under the maximum limit established by a European legislation (CE1881/2006).     

Mycotoxins in corn and wheat silage in Israel

Silage is an important feed source for intensive dairy herds worldwide. Fungal growth and mycotoxin production before and during silage storage is a well-known phenomenon, resulting in reduced nutritional value and a possible risk factor for animal health. With this in mind, a survey was conducted to determine for the first time the occurrence of mycotoxins in corn and wheat silage in Israel. A total of 30 corn and wheat silage samples were collected from many sources and analysed using a multi-mycotoxin method based on LC-MS/MS. Most mycotoxins recorded in the present study have not been reported before in Israel. Overall, 23 mycotoxins were found in corn silage; while wheat silage showed a similar pattern of mycotoxin occurrence comprising 20 mycotoxins. The most common post-harvest mycotoxins produced by the Penicillium roqueforti complex were not found in any tested samples, indicative of high-quality preparation and use of silage. Moreover, none of the European Union-regulated mycotoxins – aflatoxin B1, ochratoxin, T-2 toxin, diacetoxyscirpenol and deoxynivalenol – were found above their limits of detection (LODs). The Alternaria mycotoxins – macrosporin, tentoxin and alternariol methyl ether – were highly prevalent in both corn and wheat silage (>80%), but at low concentrations. The most prominent (>80%) Fusarium mycotoxins in corn silage were fusaric acid, fumonisins, beauvericin, monilifomin, equisetin, zearalenone and enniatins, whereas in wheat silage only beauvericin, zearalenone and enniatins occurred in more than 80% of the samples. The high prevalence and concentration of fusaric acid (mean = 765 µg kg^–1) in Israeli corn silage indicates that this may be the toxin of highest potential concern to dairy cow performance. However, more data from different harvest years and seasons are needed in order to establish a more precise evaluation of the mycotoxin burden in Israeli silage.     

Diversity in Beauvericin and Enniatins H, I, and MK1688 by Fusarium oxysporum isolated from potato

Beauvericins and enniatins are cyclohexadepsipeptide mycotoxins that exhibit phytotoxicity and insecticidal activities. In the present study, the production of beauvericin and newly found enniatins (H, I, and MK1688) was characterized in 28 Fusarium strains isolated from potato samples in Korea. The predominant Fusarium species in potato was F. oxysporum (53.6%). Fifteen strains of F. oxysporum and two strains of other Fusarium species produced beauvericin (at concentrations from 3.1 to 743.2 microg/g) in culture on rice. Enniatins H and I were produced by 3 and 11 strains at concentrations from 33.1 to 781.3 microg/g and from 6.5 to 730.3 microg/g, respectively. Five isolates produced enniatin MK1688 at concentrations from 4.6 to 432.6 microg/g. In particular, one isolate (No. 1501) identified as F. oxysporum and two other Fusarium strains (Nos. 804 and 910) produced all of the tested toxins. These results indicate that enniatins H, I, and MK1688 and beauvericin are produced by Fusarium isolates occurring on potato. We do not know if the toxins can accumulate in the environment since it was not demonstrated.     

Structural analysis of enniatin H,I, and MK1688 and beauvericin by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and their production by Fusarium oxysporum KFCC 11363P

The molecular structures of enniatins H, I, and MK1688 and beauvericin were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS fragmentation occurred by loss of -CO after opening of the cyclic molecule to carbonyl carbon, and cleavage of the peptide and ester bonds in the molecular structure. Fusarium oxysporum KFCC 11363P was tested for its ability to produce beauvericin and enniatins H, I, and MK1688 on five cereal substrates: rice, barley, maize, wheat, and Indian millet kernels. Furthermore, optimal conditions for the production of the four mycotoxins by the Fusarium isolate were examined on maize at four temperatures (15, 20, 25, and 30°C) and at three moisture contents (10, 20, and 40%). Large amounts of beauvericin and enniatin H were present in maize cultures at 25°C (232.4 and 196.4 µg g^?1, respectively). Enniatins I and MK1688 were maximally formed at 20°C (221.5 and 180.2 µg g^?1, respectively). The optimal moisture contents for the production of enniatins H (196.4 µg g^?1) and MK1688 (165.6 µg g^?1), were 40%.     

Development and validation of an UHPLC-MS/MS method for the determination of mycotoxins in grass silages

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) multi-mycotoxin analytical method was developed to simultaneously identify and quantify 20 mycotoxins in grass silages, inclusive of mycotoxins that are currently regulated in European Union feeds. Extraction of mycotoxins from dried grass silages was performed using of a modified QuEChERS extraction employing an acidified aqueous extraction (0.1 N HCl) with no further clean-up. Following chromatographic separation, analytes were detected using a fast polarity-switching MS/MS method that allowed both positive and negative ions to be analysed from a single injection, thus the reducing time and cost of analysis. The limits of detection and quantification ranged between 3 µg kg^–1 DM (aflatoxin B₁, beauvericin and enniatin A and A₁) and 200 µg kg^–1 DM (deoxynivalenol), and between 10 µg kg^–1 DM (aflatoxin B₁, beauvericin and enniatin A₁) and 500 µg kg^–1 DM (deoxynivalenol), respectively. Inter-assay accuracy and precision ranged between 90% and 107% and between 3.9% and 15.0% CV, respectively. The accuracy of the method was assessed through the application to a range of incurred samples in an inter-laboratory study.     

Survey of maize from south-western Nigeria for zearalenone, alpha- and beta-zearalenols, fumonisin B1 and enniatins produced by Fusarium species.

A survey for the natural occurrence of Fusarium mycotoxins in maize for human consumption in four south-western states of Nigeria using High Performance Liquid Chromatography coupled with Mass Spectroscopy (HPLC/MS) showed that 93.4% of the samples were contaminated with zearalenone (ZON), alpha- and beta-zearalenols (alpha- and beta-ZOL), fumonisin B(1) (FB(1)) or enniatins (ENNs). The fractions of contaminated samples were 73% for FB(1) (mean:117 microg kg(-1), range:10-760 microg kg(-1)); 57% for ZON (mean:49 microg kg(-1), range:115-779 microg kg(-1)) and 13% for alpha-ZOL (mean: 63.6 microg kg(-1), range:32-181 microg kg(-1)), while ENNs A1, B and B(1) were present in 3, 7 and 3% of the samples respectively. There was no beta-ZOL present above the quantification limits of 50 microg kg(-1). Only the FB(1) content was significantly different at the 95% confidence level among the four states. The Fusarium species most frequently isolated from maize seeds were F. verticillioides (70%), followed by F. sporotrichioides (42%), F. graminearum (30%), F. pallidoroseum (15%), F. compactum (12%), F. proliferatum (12%), F. equiseti (9%), F. acuminatum (8%) and F. subglutinans (4%). This is the first report of the occurrence of alpha-zearalenol and enniatins in Nigerian maize.     

Occurrence of enniatins and beauvericin in 60 Chinese medicinal herbs

A total of 60 Chinese medicinal herbs were examined for contamination of the emerging Fusarium mycotoxins enniatins (ENNs) A, A1, B, B1 and beauvericin (BEA). The herbs under study are commonly used in China as both medicines and food. The dried samples of herbs were randomly collected from traditional Chinese medicine stores in Zhejiang province, China. Sample preparation was achieved by methanol extraction, followed by a simple membrane filtration step; no tedious clean-ups were involved. ENNs A, A1, B, B1 and BEA were analysed by the recently developed stable isotope dilution assays, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). With limits of detection ranging between 0.8 and 1.2 µg kg^–1 for the analytes under study, 25% of all analysed samples were contaminated with at least one of the ENNs and BEA. BEA was the most frequently detected toxin with a 20% incidence in all samples. The percentages of ENN-positive samples were lower: each single ENN was detected in 6.7–11.7% of all samples. Considering the total amounts of the five mycotoxins in single samples, values between 2.5 and 751 µg kg^–1 were found. The mean total amount in positive samples was 126 µg kg^–1. Regarding ginger, the frequent occurrence of ENNs and BEA in dried ginger could be confirmed in samples from Germany. However, in fresh ginger root the toxins were not detectable. This is the first report on the presence of ENNs and BEA in Chinese medicinal herbs.     

Short communication: Analysis of mycotoxins in Spanish milk

We surveyed the presence of 22 mycotoxins in 191 Spanish cow milk samples. Mycotoxins could be carried over from diet into animal milk and have toxic effects on human and animal health. The interaction of different mycotoxins may be additive or synergetic. Therefore, surveillance of mycotoxin co-occurrence in milk is recommended. Aflatoxins M₁, B₁, B₂, G₁, and G₂, ochratoxins A and B, nivalenol, deoxynivalenol, deepoxy-deoxynivalenol, 3- and 15-acetyldeoxynivalenol, diacetoxyscirpenol, neosolaniol, fusarenon X, T-2 and HT-2 toxins, fumonisins B₁, B₂, and B₃, sterigmatocystin, and zearalenone were analyzed. Samples were treated by liquid-liquid extraction with acidified acetonitrile, followed by an acetonitrile-water phase separation using sodium acetate. The analysis was carried out by HPLC coupled to a triple quadrupole mass spectrometer. None of the analyzed mycotoxins had a concentration level higher than their detection limit (0.05–10.1 µg/L). The aflatoxin M₁ in the samples never exceeded the level established by the European Union.     

Emerging fusarium-mycotoxins fusaproliferin, beauvericin, enniatins, and moniliformin: a review

The contamination of foods and feed with mycotoxins is a commonly known problem. Intense investigations have been conducted to study the occurrence, toxicity, and recently also the prevention and detoxification strategies of mycotoxins in human and animal food chains. Most of the studies have emphasized on "traditional" mycotoxins, such as aflatoxins, ochratoxin A, and trichothecenes. However, one of the most common grain-contaminating genus of fungi, Fusarium spp., is also capable of producing other toxic secondary metabolites - the so-called emerging mycotoxins such as fusaproliferin, beauvericin, enniatins, and moniliformin. So far, only limited data is available on these metabolites. This is not only due to their late recognition but especially the late understanding of their role as mycotoxins. This paper summarizes the existing data on the chemistry, analytical techniques, biosynthesis, production, toxicity, and occurrence data on fusaproliferin, beauvericin, enniatins, and moniliformin. Based on the available studies, attention should be paid to the studies on the distinct significance of these compounds in the human and animal food chains.     

Analysis of some breakfast cereals on the French market for their contents of ochratoxin A,citrinin and fumonisin B1: development of a method for simultaneous extraction of ochratoxin A and citrinin

Crops may be contaminated by mycotoxins which can persist in the final products. Forty-five breakfast cereals were collected in French supermarkets. Ochratoxin A (OTA) and citrinin (CIT) were simultaneously extracted by a new method based on solvent partition validated in-house. The recoveries were over 80% for CIT and OTA. Fumonisin B₁ (FB₁) was analysed by an IUPAC method. The recoveries for FB₁ ranged from 50% to 70%, depending on the matrix. The losses were located at the step of immunoaffinity clean-up.OTA was detected in 69% of the samples; 20% of them were above the EU limit of 3 μg/kg. Twenty percent contained CIT (1.5–42 μg/kg). FBs were detected, not only in cornflakes, but also in products containing oats or rice, in the range 1–1110 μg/kg. Some samples were contaminated by all three mycotoxins.     

Occurrence of emerging mycotoxins in cereals and cereal-based products from the Korean market using LC-MS/MS

The present study aimed to investigate the occurrence of emerging mycotoxins in cereals (n = 61) and cereal-based products (n = 36) collected from Korean market. First of all, using the quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction method, and ultrahigh-pressure liquid chromatography (UPLC) with triple quadruple tandem mass spectrometry (MS/MS), we developed a simple and fast method for quantitative determination of eight emerging mycotoxins including alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), beauvericin (BEA) and enniatins (ENs; ENA, ENA1, ENB and ENB1). The developed analytical method was validated in parameters of linearity, precision and accuracy. For UPLC-MS/MS analysis, the recoveries of emerging mycotoxins from spiked samples at three concentration levels ranged from 82.7% to 108.8% with RSDs between 0.4% and 14.7%. Analytical methods were applied to determine the contamination of mycotoxins in cereal and cereal-based product samples. Sixty-three of the total 97 samples were contaminated with at least one emerging mycotoxin. The maximum number of emerging mycotoxins observed in a single sample was six out of eight analytes. The highest level of contamination was detected in cereal at 70.9 μg/kg for alternariol monomethyl ether (AME). However, currently there is no international standard for emerging mycotoxins in food. Accordingly, it is necessary to establish a database of emerging mycotoxins contamination through continuous monitoring.     

An integrated sample preparation to determine coccidiostats and emerging Fusarium-mycotoxins in various poultry tissues with LC-MS/MS

The usefulness of an existing sample preparation technique used for ionophoric coccidiostats (lasalocid, monensin, salinomycin and narasin) was applied in the analysis of emerging Fusarium-mycotoxins beauvericin (BEA) and enniatins (ENNs) in poultry tissues (liver and meat). Also, maduramicin and liver as a new sample matrix was introduced. The developed methods were validated and applied for the determination of coccidiostats and BEA/ENNs in Finnish poultry tissues in 2004-2005. The validation parameters demonstrated that the integrated sample preparation technique is applicable to the parallel determination of these contaminants in poultry tissues. Of the samples analysed (276 meat and 43 liver), only trace levels of LAS, MON, SAL, NAR and MAD were detected in 7, 3, 5, 6 and 4% of the samples, respectively. Interestingly, for the first time, traces of BEA and ENNs could also be detected in animal tissues. BEA and ENNs A, A1, B and B1 were found in 2, 0.3, 0.6, 4 and 3% of the samples, respectively. The simultaneous presence of coccidiostats and mycotoxins was detected in three turkey samples in 2004.     

Occurrence of certain mycotoxins in corn and corn‐based products and thermostability of fumonisin B1 during processing

A total of 57 samples of corn and corn‐based products collected from various districts of Egypt were analyzed for Fusarium mycotoxins (T‐2, diacetoxyscripenol (DAS( deoxynivalenol (DON) and fumonisin B₁ (FB₁) and aflatoxins. FB₁ was detected in about 80%, 53.85%, 33.3%, and 28.57% of yellow corn, corn meal, white corn, and popcorn samples, respectively. The levels of FB₁ ranged from 10 to 780 μg/kg. T‐2 and DAS were detected in 5% and 10% of yellow corn samples, respectively, and DON was detected in white corn and popcorn samples at levels of 28.8 and 10.1 μg/kg, respectively. Starch samples were found to be free from Fusarium mycotoxins. Baking balady bread at 450°C/min reduced FB₁ to 72.4% while baking Franco bread at 250°C/20 min reduced FB₁ to 57.4%. Boiling of macaroni and corn in water completely removed FB₁ from contaminated samples. On the other side, corn flakes samples were found to be contaminated with aflatoxins B₁ and G₁ at levels ranging from 6 to 10 ppm, whereas 2.9% of samples were contaminated with aflatoxin B₁ > 35 ppm and G1 > 16 ppm.