小麦和玉米中脱氧雪腐镰刀菌烯醇与雪腐镰刀菌烯醇的免疫亲和净化-高效液相色谱检测方法研究

目的建立小麦和玉米中脱氧雪腐镰刀菌烯醇与雪腐镰刀菌烯醇的免疫亲和净化-高效液相色谱检测方法。方法样品经纯水提取后,用免疫亲和柱净化,经甲醇洗脱,在C18色谱柱上等度洗脱分离,采用紫外检测器检测。结果标准曲线在0.1~2.0 mg/kg范围内线性良好。小麦基质中脱氧雪腐镰刀菌烯醇的回收率为72.8%~110.1%,精密度为3.6%~10.8%,实验室内Hor Rat值为0.24~0.48;玉米中脱氧雪腐镰刀菌烯醇的回收率为72.2%~90.6%,精密度为1.2%~5.2%,实验室内Hor Rat值为0.07~0.31。小麦基质中雪腐镰刀菌烯醇的回收率为58.9%~100.4%,精密度为3.6%~11.3%,实验室内Hor Rat值为0.23~0.63;玉米中雪腐镰刀菌烯醇的回收率为56.9%~91.9%,精密度为2.5%~7.8%,实验室内Hor Rat值为0.11~0.43。结论该方法具有灵敏度高、重现性好、操作简便、准确可靠等特点,适用于小麦和玉米中脱氧雪腐镰刀菌烯醇与雪腐镰刀菌烯醇的测定。     

脱氧雪腐镰刀烯醇时间分辨免疫法的建立

目的：利用抗脱氧雪腐镰刀烯醇多克隆抗体及其人工抗原,采用时间分辨免疫荧光技术建立间接竞争脱氧雪腐镰刀烯醇时间分辨免疫检测方法(DON-TRFIA)。方法：以DON人工抗原(DON-BSA)包被微孔板,与DON标准或样品中的DON共同竞争结合抗DON多克隆抗体,然后用稀土离子Eu3+标记羊抗兔抗体进行示踪检测,并对建立DON-TRFIA进行方法学的考核。结果：该方法的灵敏度为0.01ng/ml,检测范围为0.01～100ng/ml,IC50为4.84ng/ml,批内、批间变异率均小于10%。不同样品添加回收实验表明小麦、玉米、啤酒、面包回收率分别为89.41%～123.2%、115.6%～123.6%、80.35%～118%、87%～92.45%。玉米样品检测结果表明DON-TRFIA与商品化DON-ELISA试剂盒结果高度相关,具有较好的一致性。结论：DON-TRFIA是目前已见报道文献中最灵敏的DON免疫检测法,具有很好的应用前景。     

Reduction of Moniliformin in Corn by Heat Processing

The effects of autoclaving, baking, extrusion, frying, and roasting on the stability of moniliformin (MON) in spiked (5 μg/g of MON) corn‐based food products were investigated. Roasting corn meal at 218 °C for 15 min had the most significant effect (p ≤ 0.05) on the reduction of MON (44.6%). Autoclaving creamed corn at 121 °C for 65 min resulted in only 10% reduction of MON. Reductions of MON ranging from 5.4 to 28.9% were observed when corn chips were prepared from spiked masa. MON was reduced by 42.2% when corn muffins were baked and by 26.7% when corn grits were extruded. Overall, MON showed heat stability similar to or greater than other Fusarium mycotoxins such as deoxynivalenol and fumonisin B₁     

A survey of free and conjugated deoxynivalenol in the 2008 corn crop in Ontario, Canada

BACKGROUND: Deoxynivalenol (DON, vomitoxin), one of the most important mycotoxins produced by many Fusarium species, is found as a common contaminant of crops worldwide. Recent studies have described the presence of conjugated forms of DON (glycosides and fatty acid). The aim of the current study was therefore to investigate the natural occurrence of free and conjugated DON in Canadian corn. RESULTS: Free and conjugated DON was determined by gas chromatography‐mass spectrometry (GC‐MS) and enzyme‐linked immunosorbent assay (ELISA) in 86 corn samples collected from the 2008 crop in Ontario, Canada. Free DON concentrations determined by ELISA were similar to values determined in most samples using GC‐MS. Conjugated DON was detected in 72 samples. Levels of free DON ranged from 0.17 to 14.00 µg g^?1 using GC‐MS. The highest levels of free DON were found in corn samples from the southern and southwestern regions of Ontario, while samples from eastern regions were less contaminated. Conjugated DON was found mainly in corn from the east‐central region, with five of six samples showing high levels of conjugated DON (up to 43% increase in DON following acid hydrolysis). Low levels of conjugated DON (≤10% increase in DON following acid hydrolysis) were detected in the majority of corn samples from the southwestern region (nine of 19 samples) and from the central region (16 of 36 samples). CONCLUSION: The current survey emphasizes the frequency of conjugated DON in Ontario grown corn and the potential challenges in understanding the hazard posed by DON‐contaminated foodstuffs and feedstuffs. Copyright © 2011 Society of Chemical Industry     

Mycotoxins in fuel ethanol co‐products derived from maize: a mass balance for deoxynivalenol

BACKGROUND: Three matrices—corn (maize) meal, distiller's dried grains with solubles (DDGS) and condensed distiller's solubles (CDS)—were sampled in sequence from a continuous dry‐milling process plant for the determination of mass balance of deoxynivalenol (DON). Four commercially available enzyme‐linked immunosorbent assay (ELISA) kits were evaluated for their ability to measure the presence of DON. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used as standard method to detect DON and other Fusarium toxins. RESULTS: The concentrations of DON in DDGS and especially CDS were overestimated or underestimated by ELISA. However, for both matrices, all ELISA methods were not significantly different in their mean results from the LC/MS/MS standard, although the variability in results was much higher. DON concentrations in the CDS and the final DDGS co‐product were significantly higher (P ≤ 0.01) than in the starting material (corn grain). Toxin concentration increased by a factor of 3 on a dry weight basis in DDGS compared with the starting corn and by a factor of 4 in CDS. Mean concentration of DON in CDS was four times higher (7.11 mg kg^?1) than in corn grains (1.80 mg kg^?1) and 1.4 times higher than in DDGS (5.24 mg kg^?1). Mass balance calculations showed that CDS was the main source of contamination of DON, comprising ca 70% of the toxin found in the final product (DDGS). Most DON (87%) was accounted for by this analysis. CONCLUSION: Concentrations in the grain corn entering ethanol plants should be close to the dietary values recommended for swine in Canada and the USA for DON (1 mg kg^?1). Small amounts of acetyldeoxynivalenol and DON glucoside were also found in the three matrices along with a small amount of zearalenone. Unlike the situation for DON, the DON glucoside was not concentrated into DDGS and CDS, indicating that it was hydrolysed during the fermentation process. Copyright © 2009 Society of Chemical Industry     

婴幼儿谷类辅助食品加工工艺对其脱氧雪腐镰刀菌烯醇及衍生物影响

脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)是由镰刀菌在侵染小麦等禾谷类作物过程中产生的一种有毒次级代谢产物,主要污染小麦、大麦、燕麦、黑麦和玉米等谷物。DON在植物和微生物作用下会转化形成各种衍生物,且这些衍生物可与DON原型同时存在,增加了谷物及其相关制品的安全风险。谷类辅食是婴幼儿膳食中的重要组成部分,且婴幼儿对毒素的敏感性更强,应严格限制DON及其衍生物在谷类辅食中被检出。已有研究表明谷物类食品的制作加工方式会对DON及其衍生物的含量水平产生影响,但专门针对婴幼儿消费人群的谷类辅助食品的加工过程如何影响DON及其衍生物水平还鲜有报道。目前婴幼儿谷类辅助食品中DON及其衍生物含量存在超标问题,严重影响婴幼儿这类特殊人群的生长发育和健康。本文从婴幼儿谷类辅食中DON及其衍生物的污染状况及其在加工过程中的含量变化等方面进行较为详细的阐述,以期为婴幼儿谷类辅食中DON的风险评估及防控策略的制定提供有利参考。     

Simultaneous detection of deoxynivalenol and zearalenone by dual-label time-resolved fluorescence immunoassay

BACKGROUND: The health risks of deoxynivalenol (DON) and zearalenone (ZEN) necessitate the development of analytical methods for widespread food and feed screening. We sought to establish a rapid, economic and sensitive dual‐label time‐resolved fluoroimmunoassay (TRFIA) to detect DON and ZEN simultaneously. Eu³⁺‐ and Sm³⁺‐labelled antibodies were used, as lanthanides are more stable and have narrower emission spectra than most fluorescent dyes. RESULTS: The limit of detection was 0.0194 ng mL^?1 for DON (range: 0.0194–100 ng mL^?1) and 0.37 ng mL^?1 for ZEN (range: 0.37–50 ng mL^?1). DON recovery in spiked cereal samples was 88–107%, and for ZEN was 83–108%, with both intra‐ and inter‐assay coefficients of variation (CVs) less than 5%. The dual‐label TRFIA results correlated well with ELISA results (correlation coefficients: 0.9733 for DON and 0.9784 for ZEN). CONCLUSION: The dual‐label TRFIA is a simple, fast and sensitive method for high‐throughput screening of DON and ZEN in food and feedstuff. Copyright © 2010 Society of Chemical Industry     

Signal amplification using colloidal gold in a biolayer interferometry-based immunosensor for the mycotoxin deoxynivalenol

Deoxynivalenol (DON) is a toxin produced by certain species of Fusarium fungi that can infest wheat, barley and corn. The fungi cause diseases in crops worldwide and some of the secondary metabolites, such as DON, can adversely affect animal health and food safety. To monitor DON in wheat rapidly, a biosensor using the principle of biolayer interferometry (BLI) was developed. The signal from the sensor was substantially amplified through the use of a primary antibody-colloidal gold conjugate. The amplification was much greater in the presence of wheat matrix than in buffered solution, suggesting matrix components may have contributed to the enhancement. The improved signal provided by the amplification allowed for the development of rapid qualitative and quantitative assays. The limit of detection of the method was 0.09 mg kg(-1); the limit of quantitation was 0.35 mg kg(-1). Recovery from wheat spiked over the range from 0.2 to 5 mg kg(-1) averaged 103% (RSD = 12%). The quantitative assay compared favourably (r(2) = 0.9698) with a reference chromatographic method for 40 naturally contaminated wheats. The qualitative assay was able to classify accurately the same group of 40 samples as either above or below a 0.5 mg kg(-1) threshold. These results suggest that the BLI technique can be used to measure DON in wheat rapidly.     

2020年我国粮食及其产品中脱氧雪腐镰刀菌烯醇毒素污染情况与分布特征

摘要：目的 本研究旨在了解脱氧雪腐镰刀菌烯醇(Deoxynivalenol, DON)毒素在各种粮食和在全国各地区的污染情况及分布规律。方法 将粮食样品充分研磨,采用ELISA酶联免疫吸附测定法进行检测,在包被了DON毒素的微孔中加入标本,标准品和抗体。温育并洗涤之后,用酶标仪测定吸光度,计算样品浓度。 结果 在不同种类的粮食中,玉米中DON毒素含量与大米、绿豆、红豆相比差异有意义(p＜0.05)。玉米不同加工产品中的毒素含量玉米面与玉米渣,玉米与玉米渣之间有显著性差异(p＜0.01)。玉米DON毒素含量西北与北方,西北与南方比较差别有意义(p＜0.05);面粉DON含量北方与南方比较差别有意义(p＜0.05);大米中DON含量西北与南方,北方与南方比较差别有显著性意义(p＜0.01)。 结论 不同粮食毒素的含量不同,玉米中的毒素含量较高。玉米不同加工方式生产的产品毒素的含量有所不同。粮食中DON毒素含量的分布情况与环境气候有较大关系。     

Improved methodology for the simultaneous detection of the trichothecene mycotoxins deoxynivalenol and nivalenol in cereals

A simple and sensitive method has been developed for the analysis of two trichothecene mycotoxins (deoxynivalenol and nivalenol) in cereals. These toxins were extracted with acetonitrile/water (3:1), defatted with n-hexane and purified by a two-step chromatographic procedure using Florisil and Sep-pak columns. The amounts of deoxynivalenol (DON) and nivalenol (NIV) in the column eluates were quantitated by gas chromatography with electron capture detector and gas chromatography-mass spectrometry (selected ion monitoring). The limits of detection of the method were 2.0 micrograms/kg for DON and NIV with recoveries of the toxins spiked into polished rice, wheat and corn at 300 micrograms/kg averaging 87% and 86% respectively.     

脱氧雪腐镰刀菌烯醇(DON)直接竞争ELISA方法的建立

利用前期制备得到的DON酶标抗原(辣根过氧化物酶标记)以及抗DON抗体,建立检测食品中DON含量的直接竞争ELISA方法。该方法的检测范围1～100ng/mL,灵敏度达0.56ng/mL,半数抑制浓度IC50为10ng/mL;与DON类似物T-2毒素的交叉反应率为12%;玉米淀粉样品回收率在80.2%～91.1%之间,平均批间变异<15%,平均批内变异<3%。     

Signal amplification using colloidal gold in a biolayer interferometry-based immunosensor for the mycotoxin deoxynivalenol

Deoxynivalenol (DON) is a toxin produced by certain species of Fusarium fungi that can infest wheat, barley and corn. The fungi cause diseases in crops worldwide and some of the secondary metabolites, such as DON, can adversely affect animal health and food safety. To monitor DON in wheat rapidly, a biosensor using the principle of biolayer interferometry (BLI) was developed. The signal from the sensor was substantially amplified through the use of a primary antibody–colloidal gold conjugate. The amplification was much greater in the presence of wheat matrix than in buffered solution, suggesting matrix components may have contributed to the enhancement. The improved signal provided by the amplification allowed for the development of rapid qualitative and quantitative assays. The limit of detection of the method was 0.09?mg?kg^?1; the limit of quantitation was 0.35?mg?kg^?1. Recovery from wheat spiked over the range from 0.2 to 5?mg?kg^?1 averaged 103% (RSD?=?12%). The quantitative assay compared favourably (r ^2?=?0.9698) with a reference chromatographic method for 40 naturally contaminated wheats. The qualitative assay was able to classify accurately the same group of 40 samples as either above or below a 0.5?mg?kg^?1 threshold. These results suggest that the BLI technique can be used to measure DON in wheat rapidly.     

Detection of melamine using commercial enzyme-linked immunosorbent assay technology

Recent cases of adulteration with melamine have led to the need for rapid and reliable screening methods. To meet this need, commercial enzyme-linked immunosorbent assay (ELISA) test kits for the detection of triazines were evaluated. The recently released Melamine Plate kit (Abraxis, Warminster, Pa.) displayed a limit of detection of 9 ng/ml for melamine in phosphate-buffered saline (PBS) and approximately 1 microg/ml for melamine added to dog food. An atrazine ELISA test kit produced by Abraxis required 0.2 mg/ml to generate a response more than four times the standard deviation from background. In contrast, with the EnviroGard Triazine Plate kit (Strategic Diagnostics, Inc., Newark, Del.), 1.5 mg/ml melamine in PBS generated a signal only one standard deviation from background, which was insufficient to define a limit of detection. Extraction based on dilution with 105 mM sodium phosphate/75 mM NaCl/2.5% nonfat milk/0.05% Tween 20 (UD) enabled detection of fivefold less melamine in dog food than did use of the procedure recommended by the manufacturer, which entailed extraction into 60% methanol, sonication, centrifugation, filtration, and further dilution into 10% methanol/PBS. Using the Abraxis Melamine ELISA, both extraction protocols yielded identical results with a dog food sample adulterated with melamine. The recovery of melamine spiked into gravy from dog food using UD was 74% +/- 4%. In conclusion, the recently released Abraxis ELISA for melamine proved to be a useful alternative to more cumbersome methods.     

Quantitation of soya protein by enzyme linked immunosorbent assay of its characteristic peptide

An enzyme linked immunosorbent assay (ELISA) is described for the quantitative measurement of soya proteins in processed food products. The peptide fragment bearing an antigenicity against anti-glycinin (soya bean 11S globulin) antibody was purified from the trypsin digests of autoclaved glycinin and used as an indicator antigen for soya proteins. ELISA samples were prepared also by a combination of autoclaving and tryptic digestion. Quantitation of soya proteins by ELISA was found to resist interference by other food materials, and the ELISA signal was independent of soya bean cultivar. The ELISA method was evaluated on pork sausages which were prepared in the laboratory to contain 0 to 20·8% soya protein isolate. With commercial soya protein isolate as reference standard, the results showed that the detected amounts were in quantitative agreement with the added amounts of soya protein isolate.     

基于能力验证活动对玉米真菌毒素快检技术的评价

玉米原粮贸易过程中,真菌毒素快检是主要的卫生指标检测技术之一,但由于真菌毒素在原粮中的污染通常分布极不均匀,造成了检测数据差异大,准确性难于评估等问题。本研究通过广泛筛查玉米原粮天然真菌毒素污染物,研制满足均匀性、稳定性要求的玉米中脱氧雪腐镰刀菌烯醇（DON）、玉米赤霉烯酮（ZEN）质量控制样品。通过组织行业内企业参加原粮检测化验室能力验证活动,评价粮食中真菌毒素快检技术的应用情况。本研究报名参加毒素快检能力验证共153家企业,其中仓储粮库占36%、饲料养殖企业占64%。实验室遍布17个省、市、自治区,均采用食品快速检验技术检测并报送数据。DON及ZEN能力验证稳健CV分别为26%及29%,一定程度上反应了原粮毒素快检的实际使用情况,为提升快检技术及实验室原粮检测能力,提供行业数据支持。     

Development of a Simultaneous Lateral Flow Strip Test for the Rapid and Simple Detection of Deoxynivalenol and Zearalenone

The objective of this study was to develop a 1‐step simultaneous lateral flow strip test for the rapid and simple detection of deoxynivalenol (DON) and zearalenone (ZEA) in grains. Two monoclonal antibodies (MAbs) against DON and ZEA were respectively conjugated with gold nanoparticles and used to develop a lateral flow strip test for a single toxin and multiple toxins. First, individual lateral flow strips for a single toxin were optimized, and their conditions were used to develop a simultaneous lateral flow strip for multiple toxins. Limits of detection of both lateral flow strip tests for DON and ZEA were the same (DON: 50 ng/mL, ZEA: 1 ng/mL). Both methods showed cross‐reactivity for α‐zearalenol and β‐zearalenol, but no cross‐reaction to other mycotoxins. The results can be completed obtained within 15 min. The cut‐off values of the simultaneous lateral flow strip for the spiked rice and corn were 500 and 10 ng/g for DON and ZEA, respectively. The results demonstrated that the developed simultaneous lateral flow strip test offers a rapid, easy‐to‐use, and portable analytical system and can be used as a convenient qualitative tool for the on‐site detection of DON and ZEA in food and agricultural commodities.     

International interlaboratory study for the determination of the Fusarium mycotoxins zearalenone and deoxynivalenol in agricultural commodities

Twenty-eight laboratories from 12 different countries participated in an interlaboratory study for the determination of the Fusarium mycotoxin zearalenone (ZON) in maize and deoxynivalenol (DON) in maize and wheat employing their usual in-house methods. The aim of this study was to obtain information about the state-of-the-art of ZON and DON analysis in cereals and to support a knowledge and experience exchange between the participating laboratories in the field of mycotoxin analysis. Eight different sample types were distributed to the participants, 'blank' materials, spiked samples (102 mu g/kg ZON in maize and 475 mu g/kg DON in wheat) and naturally-contaminated maize and wheat. For the final separation and quantification either gas chromatography (GC), high performance liquid chromatography (HPLC), thin layer chromatography (TLC) or enzyme linked immunosorbent assays (ELISA) were employed by the participating laboratories. Coefficients of variation (CV) between laboratory mean results (outliers rejected) ranged from 28 to 41% for ZON and from 32 to 38% for DON. The results are close to the between laboratory CV criteria of 40% for DON and ZON at concentration levels of > 100 mu g/kg established by the CEN in 1999. A good trueness was obtained for the wheat samples spiked at 475 mu g/kg DON. However, a significant deviation at p = 0.01 from the respective target value was observed for the maize samples spiked at 102 mu g/kg ZON. The high CVs can be traced back to problems occurring by determination of the concentration of the participants' own calibrant solutions. Additionally, the variability of the results is strongly influenced by the use of different final separation and quantification procedures.     

酶联免疫法检测呕吐毒素的方法研究

目的:利用抗呕吐毒素的特异性单克隆抗体和包被抗原,建立间接竞争酶联免疫法(ELISA)来检测谷物及其制品中呕吐毒素含量。方法:通过方阵滴定实验确定呕吐毒素最佳抗体浓度和最佳包被抗原浓度,应用酶联免疫测试盒对谷物及其制品中的呕吐毒素进行定量检测的方法学评价,来确立该方法的各项技术指标。结果:方法的最低检测浓度10μg/L;平均RSD为5.8%;回收率平均值为104%;用该方法研制的测试盒在4℃环境下可稳定6个月以上;检测时间小于2h;结论:该方法简便、安全、灵敏度高、重复性好、特异性强、能定性和定量检测谷物及其制品中的呕吐毒素含量。     

毛细管气相色谱法测定小麦和玉米中脱氧雪腐镰刀菌烯醇

本文采用毛细管气相色谱法测定了小麦和玉米中脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)的含量,小麦、玉米样品分别用乙腈-水(84:16,V/V)提取,小麦样品提取液用硅镁型吸附剂净化,玉米样品提取液用硅镁型吸附剂和活性炭二步净化,采用N-七氟丁酰咪唑(HFBI)为衍生剂,带电子捕获检测器的GC进行检测。DON加标量为0．5～1．5mg/kg时,5次重复实验的平均回收率:小麦样品为82．2%～98．5%,玉米样品为86．0%～103．4%,相对标准偏差(RSD)分别为6．2%～7．6%和6．4%～8．0%,该法DON的最低检出限为0．01mg/kg,线性范围为0．075～15mg/k8。用该法对21个小麦样品与20个玉米样品中DON的含量进行检测,其中小麦样品中DON含量为0．153～4．618mg/kg,污染率为47．6%,超标率为9．5%;而玉米样品中DON含量为0．116～3．004mg/kg,污染率为85%,超标率为20%。     

Novel Products from Underutilized Fish Using Combined Processing Technology

Mincing or surimi-processing, followed by fermentation, extrusion and intermediate moisture food (IMF) processing were combined to investigate new ways to create fish protein based snack foods. A dough of fermented fish (minced or surimi), wheat flour, corn starch, and water was extruded and subsequently dried resulting in an IMF product at pH 5.2, water activity 0.90 and moisture about 30%. The products had a chewy texture, were shelf-stable and could be processed into flavored, high-protein snack foods.