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.     

免疫亲和层析净化-高效液相色谱法同时检测几种食品中黄曲霉毒素和赭曲霉毒素A

建立一种花生食品的前处理方法,通过高效液相色谱法检测黄曲霉毒素B1、B2、G1、G2和赭曲霉毒素A。样品经过甲醇-水提取,提取液经过滤、稀释后,滤液经过含有黄曲霉毒素和赭曲霉毒素特异抗体的免疫亲和层析净化,此抗体对黄曲霉毒素B1、B2、G1、G2、赭曲霉毒素A具有专一性,黄曲霉毒素、赭曲霉毒素交联在层析介质中的抗体上。用水将免疫亲和柱上杂质除去,用甲醇通过免疫亲和层析柱洗脱,洗脱液通过带荧光检测器的高效液相色谱仪,柱后碘溶液衍生测定黄曲霉毒素的含量;洗脱液通过带DAD检测器的高效液相色谱仪测定赭曲霉毒素的含量。本方法检出花生中黄曲霉毒素G1、B1和赭曲霉毒素A的检出限均为0.5μg/kg。,黄曲霉毒素B2、G2的检出限均为0.175μg/kg。结果表明利用免疫亲和层析净化-高效液相色谱法检测花生中的黄曲霉毒素B1、B2、G1、G2和赭曲霉毒素A,方法准确、可靠。     

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.     

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。     

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.     

Determination of aflatoxin M1 in milk by triple quadrupole liquid chromatography-tandem mass spectrometry

A sensitive and reliable method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an electrospray-positive ionization method was developed for the determination of aflatoxin M₁ in milk. This method includes simple extraction of the sample with acetonitrile by ultrasonic, separation on an MGIII-C₁₈ column using 0.01% formic acid buffer/acetonitrile (60 : 40, v/v) as mobile phase, and MS/MS detection using multiple reaction-monitoring mode. Average recoveries of aflatoxin M₁ from spiked samples at concentrations of 0.02 and 1 ng ml^?1 ranged from 77% to 94%, with a 6% relative standard deviation. The limit of detection and limit of quantification were 0.006 and 0.02 ng ml^?1, respectively. The standard curve was linear between 0.02 and 20.0 ng ml^?1. The recommended method is simple, rapid, specific and reliable for the routine monitoring of aflatoxin M₁ in milk.     

Aflatoxin levels and exposure assessment of Spanish infant cereals

Aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂) are immunosuppressant, mutagenic, teratogenic and carcinogenic agents with a widespread presence in foodstuffs. Since human exposure to aflatoxins occurs primarily by contaminated food intake, and given the greater susceptibility of infants to their adverse effects, the quantification of these mycotoxins in infant food based on cereals is of relevance. Aflatoxin levels were determined in 91 Spanish infant cereals classified in terms of non- and organically produced and several types from 10 different manufacturers, using a extraction procedure followed by inmunoaffinity column clean-up step and HPLC with fluorescence detection (FLD) and post-column derivatisation (Kobra Cell system). Daily aflatoxin intake was also assessed. Preliminary analysis showed a valuable incidence of detected infant cereal samples at an upper concentration level than the detection limit for total aflatoxin (66%), corresponding to a 46, 40, 34 and 11% for AFB₁, AFB₂, AFG₁ and AFG₂, respectively. Lower aflatoxin values (median, Q₁, Q₃) in conventional infant cereal (n?=?74, AFB₁: <LOD (n.d.; 0.02), AFB₂: n.d. (n.d.; 0.01), AFG₁: <LOD (n.d.; 0.004), and AFG₂: n.d. (n.d.; <LOD) and total AF (AFtotal): 0.01 (<LOD; 0.04 µg?kg^?1) in comparison with infant cereal ecologically produced (n?=?17, AFB₁: 0.02 (0.02; 0.21), AFB₂: n.d. (n.d.; 0.03), AFG₁: 0.02 (0.01; 0.05), and AFG₂: 0.007 (n.d.; 0.02) and AFtotal: 0.05 (0.03; 0.31 µg?kg^?1) were found. In addition, five organic formulations (3.11, 1.98, 0.94, 0.47 and 0.21 µg?kg^?1) exceeded European AFB₁ legislation (0.10?µg?kg^?1) versus two conventional cereals (0.35 and 0.12 µg?kg^?1). According to the type of infant cereal, those with cocoa had the highest aflatoxin levels. Gluten‐free and cereals with dehydrated fruits had an intermediate level and milk- or honey-based cereals and multi-cereals contained the lowest levels. With the exception of the non-compliant cocoa-based organic formulation, none of the infant cereals analyzed gave a higher intake of 1?ng?kg^?1 body weight per day, suggesting that infants fed on infant cereals are exposed to a low health hazard. Nevertheless, manufacturers are advised for continued efforts in routine monitoring and a more careful selection of raw material to minimize aflatoxin levels in these infant foods.     

2015年我国部分地区市售食用植物油中黄曲霉毒素污染调查

目的 对我国8省738份市售食用植物油中4种黄曲霉毒素(AFB₁、AFB₂、AFG₁和AFG₂)污染状况进行调查。方法 首先用酶联免疫吸附(ELISA)法快速筛检市售食用植物油中黄曲霉毒素,再用超高效液相色谱(UPLC)法对阳性样品中4种黄曲霉毒素的含量进一步确证。结果 食用植物油样品中黄曲霉毒素总量的含量范围在0.06～221.00 μg/kg之间,平均为19.30 μg/kg。4种黄曲霉毒素的污染以AFB₁为主,其检出率为17.21%(127/738),平均含量为16.20 μg/kg。其次是AFB₂、AFG₁和AFG₂。花生油中黄曲霉毒素污染较重,AFB₁超标率为11.57%(25/216)。来自广西的植物油样品中黄曲霉毒素污染较重,AFB₁超标率达19.23%(20/104)。此外,散装植物油中黄曲霉毒素污染含量高于定型包装样品。95.45%(126/132)的阳性样品检出2种或2种以上的黄曲霉毒素。结论 我国食用植物油存在黄曲霉毒素协同污染现象,并以AFB₁为主。花生油、来自广西的植物油样品以及散装食用植物油样品中黄曲霉毒素污染较高,需重点监测并加以监管。     

Distribution of aflatoxins in shelling and milling fractions of naturally contaminated rice

The objective of this study was to determine the distribution of an economically important class of mycotoxins, the aflatoxins, in rice milling fractions. Rice plants grown under field production conditions are frequently infected with types of pathogenic fungi that produce toxic metabolites (mycotoxins). Paddy (seeds) rice from healthy plants in the field was collected and stored on a farm under humid, poorly ventilated conditions. Samples were milled into four fractions (hulls, brown rice, bran and white rice) and analysed for aflatoxins (B₁, B₂, G₁ and G₂) using a validated method. Rice fractions from healthy plants, which contained low levels of aflatoxins (less than 1?µg?kg^?1), were used to determine the efficiency of the extraction method. Seeds stored under poor conditions were found to be contaminated with aflatoxins B₁ and B₂ as were the fractions. The sums of AFB₁ and AFB₂ in stored paddy rice, hulls, brown rice, bran and white rice were 141, 39, 158, 367 and 56?µg?kg^?1, respectively. The ratio of aflatoxin B₁ and B₂ was about 10?:?1. AFG₁ and AFG₂ were less than 1?µg?kg^?1. Thus, brown rice contained 92.9% of the aflatoxins in paddy rice, whereas white rice contained only 27.9%.     

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.