Detection of aflatoxins,trichothecenes, ochratoxin a and zearalenone by test strip enzyme immunoassay: A rapid method for screening cereals for mycotoxins

The development of test strip enzyme immunoassays (EIA) for the rapid detection of a number of mycotoxins is described. Monoclonal or polyclonal antibodies against aflatoxin B₁ (AFB₁), aflatoxin M₁ (AFM₁), ochratoxin A (OA), T‐2 toxin, diacetoxyscirpenol (DAS), 3‐acetyldeoxynivalenol (3‐AcDON), roridin A (RA) and zearalenone (ZEA) were immobilized on a nylon membrane. Using the corresponding toxin‐horseradish peroxidase conjugate in a direct competitive assay, dot colour development of toxinpositive test strips was visually discernible from that of the negative control. The results of the visual evaluation were compared with that of instrumental reflectance measurements of the test strips. The visual detection limits of the test strip assays for mycotoxins were at 0.6 ng ml^?1 (AFB₁) 0.2 ng ml^?1 (AFM₁), 2.0 ng ml^?1 (OA), 1.0 ng ml^?1(T‐2 toxin), 0.2 ng ml^?1 (DAS), 10.0 ng ml^?1 (3‐AcDON), 15.0 ng ml^?1 (RA), and 5.0 ppb (ZEA) respectively. Utilizing a simple extraction procedure, AFB₁, OA, T‐2 toxin, and ZEA in spiked corn samples were detected by test strip EIA at levels of 15 ng g^?1, 100 ng g^?1, 20 ng g^?1 and 80 ng g^?1 respectively.     

干燥方式对土鳖虫黄曲霉毒素含量的影响

目的 考察比较干燥方式对土鳖虫黄曲霉毒素含量的影响。方法 采购活土鳖虫,沸水烫死后,模拟产地晒干的条件设置一组样品30℃烘干,另一组样品设置为较高温度60℃快速烘干（每组各制备2批）;依据药典,采用HPLC （荧光检测器）检测比较2组样品的黄曲霉毒素含量。结果 30℃缓慢烘干者有明显腥臭味,黄曲霉毒素含量严重超标;60℃快速烘干者微有腥气,黄曲霉毒素含量合格。与传统日晒品比较,供应商大批量65℃烘干12 h者黄曲霉毒素含量合格。结论 采用较高温度及时干燥,可以控制土鳖虫黄曲霉毒素含量。     

Development of an Extraction Method of Aflatoxins and Ochratoxin A from Oral,Gastric and Intestinal Phases of Digested Bread by In Vitro Model

Validated extraction methods from in vitro digestion phases are necessary to obtain a suitable bioaccessibility study of mycotoxins in bakery products. The bakery industry produces bread with different ingredients to enrich the nutritional properties of this product and protect it from fungal growth. This bread can be contaminated by AFB₁, AFB₂, AFG₁, AFG₂ and OTA, so an extraction method was developed to analyse these five legislated mycotoxins in digested phases of two types of bread, one with wheat and the other with wheat and also enriched with Cucurbita Maxima Pepo at 20%. The studied “in vitro” digestion model consists of oral, gastric and duodenal phases, each one with different salt solutions and enzymes, that can affect the extraction and most probably the stability of the mycotoxins. The proposed method is a liquid–liquid extraction using ethyl acetate by extract concentration. These analytes and components have an important effect on the matrix effect (MEs) in the analytical equipment, therefore, validating the method and obtaining high sensitivity will be suitable. In the proposed method, the highest MEs were observed in the oral phase of digested pumpkin bread (29 to 15.9 %). Regarding the accuracy, the recoveries were above 83% in the digested duodenal wheat bread and above 76 % in the digested duodenal pumpkin wheat bread. The developed method is a rapid, easy and optimal option to apply to oral, gastric and duodenal phases of digested bread contaminated at a level of established maximum levels by European legislation (RC. 1881/2006) for food.     

深圳粮油食品中4种黄曲霉毒素联合污染状况

目的调查分析深圳地区市售粮食和食用油中4种黄曲霉毒素(AFs)的污染状况。方法以分层随机抽样得到的238份粮油食品作为研究对象,采用免疫亲和层析净化超高效色谱法测定AFB1、AFB2、AFG1和AFG24种黄曲霉毒素含量。结果大米、米制品、小麦粉、玉米粉、食用油中总黄曲霉毒素阳性率分别为35.3%、33.8%、13.9%、46.7%和24.5%。其中定型包装大米AFs阳性率(26.5%)与散装大米AFs阳性率(56.3%)差异有统计学意义(χ2=11.6,P<0.05);长江以北地区出产的大米AFs阳性率(27.3%)与长江以南地区出产的大米AFs阳性率(41.4%)差异有统计学意义(χ2=7.257,P<0.05)。米制品、小麦粉和玉米粉仅检出黄曲霉毒素B1和B2。食用油中AFB1、AFB2、AFG1和AFG2阳性率分别为24.5%、24.5%、11.3%和3.8%。AFB1超标率为5.66%,超标样本全部为作坊生产的无牌散装自榨花生油。结论深圳市售大米、食用油存在4种黄曲霉毒素联合污染的状况,污染以黄曲霉毒素B1和B2为主。深圳市售南北两地产大米污染状况有差异,散装包装大米污染状况有差异。     

免疫亲和柱-HPLC柱后光化学衍生法测定中药饮片中黄曲霉毒素B1、B2、G1、G2的含量

目的：建立中药饮片中黄曲霉毒素（ AF）B1、B2、G1、G2含量测定的方法。方法：收集中药饮片87批,用免疫亲和柱进行AF的分离处理,采用高液相色谱（ HPLC）柱后光化学衍生化法测定中药饮片中AFB1、AFB2、AFG1、AFG2含量并进行方法学分析。结果：被测定的AFB1、AFB2、AFG1、AFG2的检出限分别为0.7、0.3、0.6和0.3μg/kg,在选定的范围内线性关系良好（ r≥0.999）,精密度试验、重复性试验、准确度试验及标准样品验证试验结果均良好,87批中药饮片均未检出黄曲霉毒素。结论：免疫亲和柱-HPLC柱后光化学衍生化法操作简便,专属性强、灵敏度高,检测中药饮片中AFB1、AFB2、AFG1及AFG2的含量准确可靠。     

黄曲霉毒素B1对非洲绿猴肾细胞的毒性效应及作用机制研究

目的探讨黄曲霉毒素B1(AFB1)对非洲绿猴肾细胞(Vero E6)的毒性效应及其作用机制。方法体外培养Vero E6细胞,不同浓度AFB1处理细胞后,采用CCK-8法检测细胞存活率;检测乳酸盐脱氢酶(LDH)释放率;利用Annexin V/PI双染、DAPI染色研究细胞死亡机制。结果 AFB1对Vero E6细胞有显著毒性损伤效应,200μmol/L AFB1处理Vero E6细胞48h后,细胞存活率不到10%。细胞LDH释放率随着AFB1浓度增加而显著升高。Annexin V/PI染色检测到细胞膜磷脂酰丝氨酸外翻。DAPI染色检测到细胞核碎裂。结论 AFB1对Vero E6细胞具有明显的毒性损伤作用,其可能通过凋亡的机制引起细胞死亡。     

Aflatoxin Contamination,Its Impact and Management Strategies: An Updated Review

Aflatoxin, a type of mycotoxin, is mostly produced by Aspergillus flavus and Aspergillus parasiticus. It is responsible for the loss of billions of dollars to the world economy, by contaminating different crops such as cotton, groundnut, maize, and chilies, and causing immense effects on the health of humans and animals. More than eighteen different types of aflatoxins have been reported to date, and among them, aflatoxins B1, B2, G1, and G2 are the most prevalent and lethal. Early detection of fungal infection plays a key role in the control of aflatoxin contamination. Therefore, different methods, including culture, chromatographic techniques, and molecular assays, are used to determine aflatoxin contamination in crops and food products. Many countries have set a maximum limit of aflatoxin contamination (2–20 ppb) in their food and agriculture commodities for human or animal consumption, and the use of different methods to combat this menace is essential. Fungal infection mostly takes place during the pre- and post-harvest stage of crops, and most of the methods to control aflatoxin are employed for the latter phase. Studies have shown that if correct measures are adopted during the crop development phase, aflatoxin contamination can be reduced by a significant level. Currently, the use of bio-pesticides is the intervention employed in many countries, whereby atoxigenic strains competitively reduce the burden of toxigenic strains in the field, thereby helping to mitigate this problem. This updated review on aflatoxins sheds light on the sources of contamination, and the on occurrence, impact, detection techniques, and management strategies, with a special emphasis on bio-pesticides to control aflatoxins.     

Simultaneous Determination of Aflatoxins and Benzo(a)pyrene in Vegetable Oils Using Humic Acid-Bonded Silica SPE HPLC–PHRED–FLD

In the present work, a rapid, accurate, and cost-effective method was developed for the simultaneous quantification of aflatoxins and benzo(a)pyrene in lipid matrices, using solid-phase extraction (SPE) via humic acid-bonded silica (HAS) sorbents, followed by high-performance liquid chromatography coupled with photochemical post-column reactor fluorescence spectroscopy (HPLC–PHRED–FLD) analysis. The major parameters of extraction efficiency and HPLC–PHRED–FLD analysis were investigated and this method was fully validated. The limits of quantification and the limits of detection were 0.05–0.30 and 0.01–0.09 µg kg⁻¹, respectively. The recoveries were 66.9%–118.4% with intra-day and inter-day precision less than 7.2%. The results of 80 oil samples from supermarkets indicated a high occurrence of BaP, and most of concentrations were within the requirements of EU and China food safety regulations. This is the first utilization of HAS–SPE HPLC–PHRED–FLD to simultaneously analyze the occurrence of aflatoxins and benzo(a)pyrene in vegetable oils.     

Aflatoxin Contamination of Red Chili Pepper From Bolivia and Peru,Countries with High Gallbladder Cancer Incidence Rates

Chilean red chili peppers contaminated with aflatoxins were reported in a previous study. If the developmentof gallbladder cancer (GBC) in Chile is associated with a high level of consumption of aflatoxin-contaminated redchili peppers, such peppers from other countries having a high GBC incidence rate may also be contaminatedwith aflatoxins. We aimed to determine whether this might be the case for red chili peppers from Bolivia andPeru. A total of 7 samples (3 from Bolivia, 4 from Peru) and 3 controls (2 from China, 1 from Japan) wereevaluated. Aflatoxins were extracted with acetonitrile:water (9:1, v/v) and eluted through an immuno-affinitycolumn. The concentrations of aflatoxins B1, B2, G1, and G2 were measured using high-performance liquidchromatography (HPLC), and then the detected aflatoxins were identified using HPLC-mass spectrometry. Insome but not all of the samples from Bolivia and Peru, aflatoxin B1 or aflatoxins B1 and B2 were detected. Inparticular, aflatoxin B1 or total aflatoxin concentrations in a Bolivian samples were above the maximum levels foraflatoxins in spices proposed by the European Commission. Red chili peppers from Bolivia and Peru consumedby populations having high GBC incidence rates would appear to be contaminated with aflatoxins. These datasuggest the possibility that a high level of consumption of aflatoxin-contaminated red chili peppers is related tothe development of GBC, and the association between the two should be confirmed by a case-control study.     

免疫亲和净化HPLC柱后光化学衍生荧光法测定动物药中黄曲霉毒素

目的采用免疫亲和净化HPLC柱后光化学衍生荧光检测法测定动物药中黄曲霉毒素,考察该方法在动物药黄曲霉毒素测定中的可行性。并对动物药中黄曲霉毒素污染情况进行筛查,为动物药的监管提供依据。方法样品经有机溶剂提取、免疫亲和柱净化后,利用高效液相色谱-光化学衍生-荧光检测器进行分析测定。对部分动物药,尤其是可能污染黄曲霉毒素的动物药进行加样回收率考察,测定动物药中黄曲霉毒素残留量,并对测定结果进行分析。结果动物药中黄曲霉毒素B1、B2、G1、G2的回收率均在70%~120%。16种64批动物药中有6种24批动物药检出了黄曲霉毒素,检出率为37.5%,其中4种13批动物药均出现了超过《中国药典》2015年版限度的现象,不合格率为20.3%。结论免疫亲和净化HPLC柱后光衍生荧光检测法适用于动物药中黄曲霉毒素的测定。动物药的个别品种黄曲霉毒素污染情况较为严重,应尽快完善动物药的质量标准,保障用药安全。