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。     

A Review on Mycotoxins in Food and Feed: Malaysia Case Study

Fungi are distributed worldwide and can be found in various foods and feedstuffs from almost every part of the world. Mycotoxins are secondary metabolites produced by some fungal species and may impose food safety risks to human health. Among all mycotoxins, aflatoxins (AFs), ochratoxin A (OTA), trichothecenes, deoxynivalenol (DON and T‐2 toxin), zearalenone (ZEN), and fumonisins (FMN) have received much attention due to high frequency and severe health effects in humans and animals. Malaysia has heavy rainfall throughout the year, high temperatures (28 to 31 °C), and high relative humidity (70% to 80% during wet seasons). Stored crops under such conditions can easily be contaminated by mycotoxin‐producing fungi. The most important mycotoxins in Malaysian foods are AFs, OTA, DON, ZEN, and FMN that can be found in peanuts, cereal grains, cocoa beans, and spices. AFs have been reported to occur in several cereal grains, feeds, nuts, and nut products consumed in Malaysia. Spices, oilseeds, milk, eggs, and herbal medicines have been reported to be contaminated with AFs (lower than the Malaysian acceptable level of 35 ng/g for total AFs). OTA, a possible human carcinogen, was reported in cereal grains, nuts, and spices in Malaysian market. ZEN was detected in Malaysian rice, oat, barley, maize meal, and wheat at different levels. DON contamination, although at low levels, was reported in rice, maize, barley, oat, wheat, and wheat‐based products in Malaysia. FMN was reported in feed and some cereal grains consumed in Malaysia. Since some food commodities are more susceptible than others to fungal growth and mycotoxin contamination, more stringent prevention and control methods are required.     

酶联免疫吸附法快速检测黄曲霉毒素

结合间接竞争反应机制,运用酶联免疫吸附技术(enzyme-linked immunosorbent assay,ELISA),从抗原(antigen,Ag)包被时间、体系反应温度、酶标二抗作用时间、显色时间等主要因素开展快速检测黄曲霉毒素改进方法的研究。研究结果表明适宜的优化条件为:采用Ag包被20 h、反应温度24℃左右、酶标二抗作用时间30 min、底物显色时间15min。通过对饲料等11种样本的检测得出:半抑制浓度IC50<0.1μg/L,添加回收率在67%~116%,灵敏度达到0.03μg/L,线性系数>0.99,板内变异小于5%。检测试验结果良好,表明该改进方法具有可行性。     

发酵豆制品潜在风险因子分析

采集了59份重庆生产酱油、豆豉、腐乳和豆瓣酱4大类发酵豆制品,检测其理化指标和可能存在的6种风险因子,包括蛋白质降解物挥发性盐基氮、硫化氢和生物胺、防腐剂苯甲酸和丙酸及微生物代谢毒素黄曲霉毒素。结果表明,各类样品的pH值及总酸含量均在正常范围;腐乳及包装酱油样品所含氨基酸态氮含量均满足国家标准;各类生物胺普遍存在于发酵豆制品中,不同类型的样品中所含生物胺的种类和含量不同,主要含有酪胺和腐胺。腐乳中生物胺总量最高;挥发性盐基氮的含量较高,可能对样品安全性有影响;硫化氢含量较少,暂不影响食品的可食性;总黄曲霉毒素的含量均未超过国家限量标准,安全性良好。苯甲酸和丙酸普遍存在于各类发酵豆制品中,个别样品中苯甲酸和丙酸含量远高于国家的限量标准,问题较为突出。有必要追踪发酵豆制品加工及贮藏过程中风险因子的变化及产生规律,提出有效的质量安全控制措施。     

黄曲霉毒素检测方法研究进展

黄曲霉毒素主要是由黄曲霉和寄生曲霉产生的一类次级代谢产物,经常污染粮油原料及其制品,具有强致癌性.介绍了黄曲霉毒素毒性及一些国家和地区的残留限量标准;针对不同基质间组分存在较大差异,概述了液液萃取、超临界流体萃取、固相萃取及免疫亲和柱净化等样品前处理方法;同时对薄层色谱、高效液相色谱、酶联免疫吸附等测定黄曲霉毒素的方法、原理、应用及特点进行了综述;最后分析了黄曲霉毒素检测技术的发展趋势.     

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.