Aflatoxin B1 and sterigmatocystin survey in beer sold in China

ABSTRACT

A total of 101 samples of beer from the Chinese market were analysed for the presence of aflatoxin B₁ (AFB₁) and sterigmatocystin (STC), using methods based on liquid chromatography–tandem mass spectrometry. The limit of quantification and the limit of detection in beer were 0.1 and 0.03 µg/kg, respectively. Recoveries of AFB₁ and STC from spiked beer samples were 97.8–103.6% and 92.7–102.1%, respectively. None of the beer purchased samples were contaminated with AFB₁ or STC.     

Aflatoxin B1 in sesame seeds and sesame products from the Greek market

Aflatoxin B₁ (AFB₁) is considered as the most potent liver carcinogen for humans. A method for determination in sesame seeds was developed. AFB₁ was extracted by methanol-water, cleaned by immunoaffinity columns and determined by high-performance liquid chromatography with fluorescence detection. The recovery factor and the limit of detection (LOD) of AFB₁ in sesame seeds were 111.5% and 0.02 ng g^?1, respectively. Thirty samples of sesame products were examined for the presence of AFB₁. After analysis, 77.6% of samples were found to be contaminated. Eight samples exceeded the European Union (EU) limit (2 µg AFB₁ kg^?1). In 15 samples, AFB₁ was below the EU limit. Seven samples remained below the LOD. The most contaminated (14.49 ng AFB₁ g^?1) sample was unpeeled packaged sesame seeds. In all samples, aflatoxigenic Aspergilli fungi as well as the risk for AFB₁ presence in sesame seed was investigated.     

Aflatoxin B1-degrading activity from Bacillus subtilis BCC 42005 isolated from fermented cereal products

ABSTRACT

Aflatoxin B₁ is a naturally occurring mycotoxin that is produced as secondary metabolite by Aspergillus spp., especially A. flavus and A. parasiticus. This is the most severe toxin due to its carcinogenic, mutagenic, and teratogenic properties. Hence, methods for toxin degradation have been received increasing interest from both scientific communities and industries. In this study, 32 isolates of Bacillus spp. from various fermented cereal products were screened for their aflatoxin B₁ degradation ability. The results indicated the extracellular fraction of Bacillus subtilis BCC 42005 isolated from Iru (African locust bean) potentially possessed aflatoxin B₁-degrading ability. The maximum activity of the active fraction was at 50°C and pH 8.0. The activity was stable in a wide range of pH (5.0–8.0) and temperature (25–60°C). The aflatoxin B₁-degrading mechanisms of this strain may be possibly involved by enzyme(s). This extracellular fraction was not toxic at IC₅₀ 4 mg/ml and it can be combined with water as a soaking agent for maize, which results in 54% of aflatoxin B₁ reduction after contact time 120 min. Hence, the extracellular fraction of Bacillus subtilis BCC 42005 can be further applied as an effective soaking agent in a pretreatment process with a practical and easy-to-implement condition and also probably used to reduce the aflatoxin B₁ contamination in other foods and feeds commodities.     

Aflatoxin M1 in raw milk and aflatoxin B1 in feed from household cows in Singida,Tanzania

Aflatoxin M₁ (AFM₁) contamination in raw milk from household cows fed with sunflower seedcakes or sunflower-based seedcake feeds was determined in 37 milk samples collected randomly from different locations in Singida region, Tanzania. Aflatoxin B₁ (AFB₁) contamination in sunflower-based seedcake feed was determined in 20 feed samples collected from the same household dairy farmers. The samples were analysed by RP-HPLC using fluorescent detection after immunoaffinity column clean-up. Recoveries were 88.0% and 94.5%, while the limits of detection (LOD) were 0.026 ng mL^?1 and 0.364 ng g^?1 for AFM₁ and AFB₁, respectively. Of the analysed cow’s milk samples, 83.8% (31/37) contained AFM₁, with levels ranging from LOD to 2.007 ng mL^?1, exceeding both the European Commission (EC) and Tanzania Food and Drug Authority (TFDA) limit of 0.05 ng mL^?1. Of the contaminated samples, 16.1% exceeded the Codex Alimentarius limit of 0.5 ng mL^?1. AFB₁ was present in 65% (13/20) of the feed samples with levels ranging from LOD to 20.47 ng g^?1, 61.53% exceeding the TFDA and EC maximum limits of 5 ng g^?1 for complete dairy animal feed. The observed AFM₁ and AFB₁ contamination necessitates the need to raise awareness to dairy farmers in Tanzania to safeguard the health of the end-users.     

Aflatoxins in dairy cow feed,raw milk and milk products from Turkey

This study aims to detect aflatoxins (AFs) in dairy cow feed, milk and milk products using a high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method. All the validation parameters met the method performance criteria of the European Union. The samples comprised 76 dairy cow feeds and 205 milk and milk products (including yoghurt and yoghurt-based beverage, ayran). AFs were present in 26.3% of the feed samples. Two feed samples exceeded the maximum limit (ML) of 5 µg kg^?1 for AFB₁ as established by the EU. Nineteen milk samples (21.1%) contained aflatoxin M₁ (AFM₁) of which three exceeded the EU ML of 0.05 µg l^?1. In addition, only two yoghurt samples and one ayran sample contained AFM₁, but the levels were lower than the EU ML.     

Activated carbons as potentially useful non-nutritive additives to prevent the effect of fumonisin B1 on sodium bentonite activity against chronic aflatoxicosis

Aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) are mycotoxins that often co-occur in feedstuffs. The ingestion of AFB₁ causes aflatoxicosis in humans and animals. Sodium bentonite (NaB), a cheap non-nutritive unselective sequestering agent incorporated in animal diets, can effectively prevent aflatoxicosis. Fumonisins are responsible for equine leukoencephalomalacia and porcine pulmonary oedema, and often have subclinical toxic effects in poultries. Fumonisin B₁ and aflatoxin B₁ are both strongly adsorbed in vitro on sodium bentonite. Co-adsorption studies, carried out with a weight ratio of FB₁ to AFB₁ that mimics the natural occurrence (200:1), showed that FB₁ greatly decreases the in vitro ability of NaB to adsorb AFB₁. The ability of two activated carbons to adsorb FB₁ was also investigated. Both carbons showed high affinity for FB₁. A complex behaviour of the FB₁ adsorption isotherms with pH was observed. In vitro results suggest that under natural contamination levels of AFB₁ and FB₁, a mixture of activated carbon and sodium bentonite might be potentially useful for prevention of sub-acute aflatoxicosis.     

Aflatoxin B1 in betel nuts (Areca catechu L.) imported to Pakistan from different regions of South Asia

Aflatoxin B₁ (AFB₁) levels were evaluated in betel nuts (Areca catechu L.) being imported to Pakistan during 2010–2011. In total, 278 betel nut samples (India = 21, Indonesia = 51, Sri-Lanka = 34 and Thailand = 172) were received from the Department of Customs and were analysed by thin layer chromatography (TLC). All Indian origin betel nuts showed AFB₁ contamination ranging from 11.7–262.0 µg kg^?1 with a mean of 92.5 µg kg^?1. Among Indonesian and Sri Lankan shipments, 80.4% and 73.5% betel nuts were contaminated with AFB₁ ranging between 3.3–39.2 and 6.5–103.4 µg kg^?1 with a mean of 11.6 and 35.0 µg kg^?1, respectively. However, only 30.2% of Thailand origin samples showed AFB₁ contamination ranging 3.3–77.0 µg kg^?1 with a mean of 6.6 µg kg^?1. The widespread occurrence of AFB₁ increases the hazard associated with betel nuts. Thus, strict control is a pre-requisite for the production and import/export of psychoactive substances as betel nuts.     

Degradation of aflatoxin B1 in peanut meal by electron beam irradiation

The effects and safety of electron beam irradiation (EBI) treatment on the detoxification of aflatoxin B₁ (AFB₁) in the peanut meal were evaluated in this article. The AFB₁ degradation was predominantly affected by both initial AFB₁ and water concentrations. The degradation of AFB₁ in the selected concentrations (0.5–5 ppm) was proven to follow pseudo first-order reaction kinetics (R² > 0.95). The AFB₁ degradation was faster when the initial concentration was 5 ppm and the moisture content was 21.47%, in comparison with the initial concentration of 1 ppm and 0.5 ppm and the moisture content of 14.32% and 8.74%, respectively. The Ames and cytotoxicity tests were employed to evaluate the residual toxicity of EBI-treated peanut meal. The mutagenic activity of EB-treated samples was completely lost compared with that of untreated samples and the degradation products in peanut meal has almost no cell toxicity.     

In vivo toxicity assessment of aflatoxin B1-contaminated corn after ozone degradation

Corn is an important food and feedstuff in China and worldwide. The problems caused by aflatoxin B₁-contaminated corn (ACC) are of great concern. Our previous studies have demonstrated that ozone can effectively degrade AFB₁ in corn, prompting us to investigate the in vivo toxicity of treated ACC. In this study, 35 Kunming mice were used to assess the in vivo toxicity of ozone treated ACC. Results indicated that compared to mice fed with basal feedstuff (provided by the Shanghai SLAC Laboratory), those fed with ACC have significantly decreased mean weight as well as total protein (TP), albumin (ALB), and globulin (GLB) contents (p < 0.05). On the other hand, the liver and kidney/body weight ratio as well as the serum alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels significantly increased (p < 0.05). Obvious histopathological changes were found in the liver and kidney. When mice were fed with the ozone-treated ACC, no significant differences were observed in the mean weight, the liver and kidney/body weight ratio and in the major serum indexes ALT, TP, ALB, and GLB (p > 0.05). However, AST and ALP significantly increased (p < 0.05), and slight histopathological changes were found in liver tissues. This study indicated that ACC may lead to significant changes in various physiological characteristics and biochemical indexes in liver and kidney tissues, but ozone treatment of ACC could significantly reduce these changes.     

Degradation of aflatoxin B1 by fungal laccase enzymes

The enzymatic degradation of aflatoxin B₁ (AFB₁) by white rot fungi through laccase production was investigated in different liquid media. A significant (P < 0.0001) correlation was observed between laccase activity and AFB₁ degradation exhibited by representatives of Peniophora and Pleurotus ostreatus cultivated in minimal salts (MSM) (r = 0.93) and mineral salts — malt extract (MSB–MEB) (r = 0.77) liquid media. Peniophora sp. SCC0152 cultured in MSB–MEB liquid medium supplemented with veratryl alcohol and sugarcane bagasse showed high laccase activity (496 U/L), as well as 40.45% AFB₁ degradation as monitored using high performance liquid chromatography. P. ostreatus St2-3 cultivated in MSM liquid medium supplemented with veratryl alcohol resulted in laccase activity of 416.39 U/L and 35.90% degradation of AFB₁. Aflatoxin B₁ was significantly (P < 0.0001) degraded when treated with pure laccase enzyme from Trametes versicolor (1 U/ml, 87.34%) and recombinant laccase produced by Aspergillus niger D15-Lcc2#3 (118 U/L, 55%). Aflatoxin B₁ degradation by laccase enzyme from T. versicolor and recombinant laccase enzyme produced by A. niger D15-Lcc2#3 coincided with significant (P < 0.001) loss of mutagenicity of AFB₁, as evaluated in the Salmonella typhimurium mutagenicity assay. The degradation of AFB₁ by white rot fungi could be an important bio-control measure to reduce the level of this mycotoxin in food commodities.     

Aflatoxin M1 in buffalo and cow milk in Afyonkarahisar,Turkey

Potential hazardous human exposure to aflatoxin M₁ (AFM₁) via consumption of milk and milk products has been demonstrated by many researchers. The aim of this study was to investigate the presence of this mycotoxin in buffalo and cow milk samples in the city of Afyonkarahisar, Turkey. For this purpose, 126 buffalo and 124 cow milk samples were collected from dairy farms in Afyonkarahisar province. AFM₁ levels were determined by high-performance liquid chromatography with tandem mass spectrometric detection. Although AFM₁ was not detected in cow milk samples, AFM₁ was found above the limit of detection (<0.008–0.032 µg/L) in 27% (34 out of 126) of the buffalo milk samples. The results of this study indicated the importance of continuous surveillance of commonly consumed milk or milk product samples for AFM₁ contamination in Turkey.     

Fungi and natural incidence of selected mycotoxins in barley rootlets

The storage of barley rootlets is increasingly employed to provide raw material for pig feeding in Brazil. Barley rootlets represent an important feedstuff for animal production due to their high levels of protein and fiber, and low price. However, poor management of raw materials during storage can result in fungal growth, the loss of nutritive substances and contamination by mycotoxins. The aims of this work were (1) to identify fungi associated with barley rootlets used as pig feedstuff raw material, and (2) to identify and quantify selected mycotoxins naturally produced by isolated mycotoxin-producing species in this substrate over a year. Samples were examined for fungal counts and genera distribution. Fumonisin B₁ and aflatoxin B₁ contamination were determined using high pressure liquid chromatography (HPLC). Barley rootlet samples were of low hygienic quality. Although a broad survey was undertaken, low fungal diversity was found. Fusarium verticillioides was the most prevalent species followed by Aspergillus flavus. Despite Aspergillus clavatus being widely associated with high-moisture sprouted grains including brewers’ grains, and causing toxicity to livestock, it was not detected in this work. Although pre-harvest contamination of the barley crop, as in the maize, could occur, the barley might support F. verticillioides/Fusarium proliferatum growth when grain is remoistened during the germination and malting process and it might even continue during storage on pig farms. All samples were positive for fumonisin B₁ whereas aflatoxin B₁ contamination was not detected. It is important to point out the potential risk of fumonisin contamination in barley rootlets used as animal feed. Fusarium toxins are important not so much for their acute effects as for the chronic syndromes reported worldwide. The obtained results reveal the need for periodic monitoring of raw materials to avoid problems in animal production and hazards to animal and human health.     

Reduction of aflatoxin B1 contamination in wheat by various cooking treatments

To investigate the effects of various cooking treatments such as washing, heating and steaming on the reduction of aflatoxin toxicity, a simultaneous analytical method for aflatoxin B₁, B₂, G₁, G₂ was established using high performance liquid chromatography (HPLC) with a fluorescence detector. The levels of aflatoxin B₁ (AFB₁) spiked in wheat—three varieties of United States (US) wheat and two varieties of Korean wheat—were analyzed according to washing time and heating temperature. Reduction of AFB₁ toxicity was directly proportional to washing time in both Korean and US wheat. The concentration of AFB₁ was reduced more by heating than washing treatment. The level of AFB₁ in dried wheat was decreased to 50% and 90% by heating at 150 and 200 °C, respectively. However, the reduction of AFB₁ in wet wheat in which water (10%) was intentionally added was higher by heating than in dried wheat. The reduction of AFB₁ was increased by 8% and 23% in 10% water-added US wheat (soft red white wheat) and Korean wheat (Anbaekmil) compared to dried US and Korean wheat, respectively, through heat treatment. Traditional processing used in Korean foods such as Sujebi (a soup with wheat flakes) and steamed bread caused 71% and 43% decrease in aflatoxin B₁ content.     

Kinetic modelling of aflatoxins B1 conversion and validation in corn,rice, and peanut during thermal treatments

A kinetic model of the aflatoxins B₁ conversion was plotted and validated successfully in the aflatoxin B₁-contaminated rice during thermal treatments. Specifically, the kinetic Model A and Model B of the aflatoxin B₁ conversion were plotted based on the differential-scanning calorimetry and thermogravimetric analysis, respectively, with the pure aflatoxin B₁. In succession, Model A and Model B were validated in the aflatoxin B₁-contaminated corn, rice, and peanut during thermal treatments. Model A successfully simulated the conversion of aflatoxin B₁ in the aflatoxin B₁-contaminated rice with the correlation coefficients of 0.859 and average-absolute deviation of 6.61. The activation energy and conversion order of the aflatoxin B₁ conversion were 89.0 kJ/mol and 0.12, respectively. Moreover, the relationships between the conversion degree and time vs temperature were plotted based on Model A. These plots would help to predict the final content of aflatoxin B₁ after thermal treatments, and give an instruction to develop a food processing.     

Aflatoxins in sunflower seeds and unrefined sunflower oils from Singida,Tanzania

A total of 61 samples comprising sunflower seeds (40) and unrefined sunflower oils (21) samples collected randomly from Singida, Tanzania were analysed by Reverse Phase-high performance liquid chromatography (RP-HPLC). 15% (6/40) of the seed samples were contaminated with aflatoxin B₁ ranging from limit of detection (LOD) to 218 ng g^?1 with three of them exceeding the European Commission/European Union (EC/EU) and Tanzania Bureau of Standards (TBS)/Tanzania Food and Drug Authority (TFDA) maximum limits of 2 ng g^?1 for AFB₁ in oilseeds. The levels of total aflatoxins (AFT) in seeds ranged from LOD to 243 ng g^?1. Other aflatoxins, except AFG₂, were also detected. For the unrefined sunflower oils, the levels of AFB₁ ranged from LOD to 2.56 ng mL^?1. About 80.9% (17/21) of the analysed oil samples contained AFB₁ of which 17.65% (3/17) exceeded the EC/EU and TBS/TFDA maximum limits of 2 ng mL^?1. Other aflatoxins were also detected in the oils. The measured levels indicate there is a need for food quality education among food processors.     

Determination of Vitamin B12 in meat products by RP-HPLC after enrichment and purification on an immunoaffinity column

A quantitative method for the determination of Vitamin B12 in meat products by RP-HPLC and UV detection was developed and compared to the reference method (microbiological assay, MBA). Vitamin B12 was extracted with 50 mM sodium acetate buffer in the presence of sodium cyanide. For the quantification of total Vitamin B12, it was necessary to release protein-bound Vitamin B12 by pepsin treatment. Cyanocobalamin was detected as total Vitamin B12 after purification and enrichment on an immunoaffinity column. The calibration with five concentrations of Vitamin B12 was linear with a regression coefficient r2 > 0.99. The method was validated at three different concentration levels (5-15 ng/g) with salami showing good recovery rates between 80 and 108% and low relative standard deviations between 1.50 and 7.26% (n = 6). The detection limit was found to be 2 ng/g. The Vitamin B12 levels of 50 meat products measured by the developed procedure were similar or significantly lower than those determined by the MBA.     

黄曲霉毒素B1检测方法的研究分析

黄曲霉毒素B1是黄曲霉毒素中毒性最强、危害最大的一种。由于国内外限量标准的不统一,在出口食品中由于黄曲霉毒素不合格而被通报的食品占了相当大的比例。这也对我国检测黄曲霉毒素B1的技术提出了更高的要求,在众多检测方法中酶联免疫法也因其快速、方便等特点,逐渐被认可,成为了黄曲霉毒素B1检测工作初筛的工具。     

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products

A sensitive and specific monoclonal antibody (Mab) against aflatoxin M₁ (AFM₁), named as 2C9, was selected by semi-solid HAT medium. It exhibited high affinity for AFM₁ of 1.74 × 10⁹ L/mol and no cross-reactivity to aflatoxin B₁, B₂, G₁ and G₂. Based on the antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM₁ in milk and infant milk products. Assays were performed in the AFM₁-BSA coated (0.0625 μg/mL) ELISA format in which the antibody was diluted 1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assay performance were optimised. Finally, the limits of detection were 3 ng/L for milk and 6 ng/L for milk-based cereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged from 91% to 110%. Thirty samples were analysed, and concordant results were obtained when the data were compared with a reference high-performance liquid chromatography method.     

酶联免疫吸附法测定云南铁皮石斛中黄曲霉毒素B1

目的建立酶联免疫吸附法测定铁皮石斛中黄曲霉毒素B_1(afatoxin B_1, AFB_1)的分析方法,并研究酶联免疫吸附法(enzyme-linkedimmunosorbentassay,ELISA)和高效液相色谱法(highperformanceliquid chromatography, HPLC)测定云南铁皮石斛中AFB_1的结果差异性。方法用甲醇+水溶液(50+50, V/V)提取石斛中AFB_1,采用酶联免疫吸附法和高效液相色谱法同时检测云南铁皮石斛(铁皮枫斗)中AFB_1,进行准确性、精密度和回收率等方法学实验,比较2种方法的差异性。结果 ELISA法在测定范围内AFB_1与其对应的吸光度值之间呈良好的线性关系,回归方程Y=-34.798X+18.134,相关系数r~2为0.9967;添加1.0、4.0、10.0μg/kg3个浓度的平均加标回收率为85.3%～94.7%,相对标准偏差为2.57%～4.88%。2种方法的结果符合率为93.2%～117%,相对标准偏差均小于10%。结论 ELISA法具有操作简单、检测速度快、灵敏、特异性好等优点,可同时检测大批量样品中AFB_1的含量。