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。     

脱氧雪腐镰刀菌烯醇ELISA定量检测试剂盒的研制

研制了脱氧雪腐镰刀菌烯醇ELISA试剂盒,用于检测谷物粮食中的脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)。在抗DON单克隆抗体的基础上,用间接竞争酶联免疫吸附检测试剂盒各项指标。结果为:试剂盒最低检测浓度为20ng/mL,检测线性范围为50～400ng/mL,IC50103ng/mL。平均加标回收率>80%,批内变异系数小于10%,批间变异系数<15%。用试剂盒测定盲样,其结果与德国拜发试剂盒及免疫亲和柱-高效液相色谱检测结果无显著性差异。说明试剂盒指标符合相关技术要求,具有快速、灵敏、准确、方便等特点。     

酶联免疫吸附法测定小麦中脱氧雪腐镰刀菌烯醇

目的 建立小麦中脱氧雪腐镰刀菌烯醇(deoxynivalenol, DON)含量测定的酶联免疫吸附(ELISA)分析方法。方法 选取山东省4地区小麦样品80份, 用不同溶剂对小麦中的DON进行提取后,构建小麦中DON的ELISA检测方法, 根据国家标准对检测结果进行评价。结果 选用84%的乙腈水溶液作为提取溶剂, 其平均回收率可达100.2%, 较为理想。该ELISA方法检测小麦中DON, 线性范围是5~5000 ng/g, 回归方程Y= 0.0419X 0.0222, 相关系数r=0.9868, 样品中DON含量为5.27~3639.38 ng/g , 中位数为79.25 ng/g, 在所检测的80份样品中, 有4份超出了国家规定的上限标准(1000 ng/g), 76份符合国家规定, 合格率为95％。结论 该方法灵敏、快捷, 适用于小麦中脱氧雪腐镰刀菌烯醇的检测。     

脱氧雪腐镰刀菌烯醇酶联免疫检测方法的建立

应用抗脱氧雪腐镰刀菌烯醇单克隆抗体12D1建立了竞争间接ELISA方法,用于检测小麦、玉米中的脱氧雪腐镰刀菌烯醇(DON),最低检出限为20ng/mL,检测范围为20￣460ng/mL。用蒸馏水提取掺合DON(250￣4000ng/g)的小麦样品,回收率为82.1%￣96.6%,变异系数为0.6%￣7.1%。     

Mycotoxins in cereal grain. Part 13. Deoxynivalenol and 3-acetyl-deoxynivalenol in wheat kernels and chaff with head fusariosis symptoms

In the period of harvest at 1987 field samples of wheat heads with fusariosis symptoms were collected in 18 provinces of Poland. Subsamples of heads infected with Fusarium culmorum (W.G.Sm) Sacc. and Fusarium graminearum Schwabe were analyzed for Fusarium metabolites. In fractions of kernels with visible Fusarium-damage deoxynivalenol (DON) was present in average amount of 18.7 mg/kg (range 9.6-25.3 mg/kg) and 3-acetyl DON 1.9 mg/kg. Fractions of kernels without symptoms of visible Fusarium-damage contained only DON at average 2 mg/kg (range 0.8-3.6 mg/kg). A method of approximate calculation of DON content in a given lot of grain is proposed which may be realized during samples grading.     

The Contamination of Kenyan Lager Beers with Fusarium Mycotoxins

Seventy five samples of two popular lager beers, namely Pilsner and Tusker were randomly collected from the city of Nairobi and the surrounding satellite towns in Kenya. The samples were analyzed for the presence of 4 mycotoxins, namely, deoxynivalenol (DON), fumonisin B₁ (FB₁), zearalenone (ZEA), and aflatoxin B₁ (AFB₁), by the competitive enzyme linked immunosorbent assay (ELISA) technique. The incidences of DON and ZEA were 100% in both brands, while for FB₁ the incidence was 72%, with incidences in Tusker (76.9%) being significantly higher than in Pilsner (66.7%) (p = 0.00). The mean values for contamination were 3.29 and 3.57 ng/mL for DON, 0.28 and 0.32 ng/mL for FB₁ and 7.84 and 8.50 pg/ml for ZEA in Tusker and Pilsner brands respectively. A positive occurrence association was found between DON and FB₁ and DON and ZEA, an indication of their common source from Fusarium sp. The results suggest low levels and safe exposure to consumers of Kenyan lager beers with Fusarium mycotoxins.     

Further survey on the Fusarium mycotoxins in Korean cereals

Fifty-one samples of cereals from the 1984 harvest from Korea were analyzed for nivalenol (NIV), fusarenon-X (FX), deoxynivalenol (DON) and 3-acetyl-DON by gas chromatography (GC) utilizing a 63Ni electron capture detector (ECD), and were quantitated for zearalenone (ZEN) by high-performance liquid chromatography (HPLC) with a fluorescence detector (FD). Trichothecenes and ZEN in the positive samples were confirmed by GC-mass spectrometry (MS). Out of 51 samples, 51, 46 and 42 were positive for NIV, DON and ZEN, respectively, and one malt sample was heavily contaminated with NIV (2675 ng/g) and DON (246 ng/g), and one wheat sample was heavily contaminated with NIV (3169 ng/g). Neither FX nor 3-acetyl-DON was detected in any of the samples. The data reported here indicates that Korean cereals harvested in 1984 are simultaneously contaminated with NIV, DON and ZEN, and the incidences and levels are similar to those observed in the cereals harvested in 1983.     

Occurrence of deoxynivalenol (vomitoxin) in processed cereals and pulses in Turkey

The aim was to investigate the occurrence of deoxynivalenol (DON) in cereal and pulse products in Turkey. DON was detected using high-performance liquid chromatography (HPLC) with ultraviolet detection at 220 nm and positive results greater or equal to 0.60 ppm were confirmed by thin layer chromatography (TLC). An acetonitrile-water (21:4 v/v) extract of the sample was cleaned up on a column packed with alumina-Celite-charcoal (0.35 + 0.25 + 0.40 g). The detection limits for DON were 3 ng/injection (0.10 ppm) and 50 ng/spot (0.60 ppm) for HPLC and TLC, respectively. Eighty-three commercially available cereal and pulse product samples collected from markets and street bazaars were analysed. The recovery rates for boiled, pounded wheat and rice spiked with added DON (1 ppm) were 80.9% (SD 8.37, n =5) and 72.3% (3.85, n =5), respectively. DON was detected in six (8.82%) of 68 cereal and in none of 15 pulse products. The maximum detected amount was 2.67 ppm in a corn flour sample.     

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%。     

Fusarium mycotoxin content of UK organic and conventional barley

Each year (2002–2005), approximately 100 samples of barley from fields of known agronomy were analysed for ten trichothecenes by gas chromatography-mass spectrometry (GC/MS) including deoxynivalenol (DON), nivalenol, 3-acetyl DON, 15-acetyl DON, fusarenone X, T-2 toxin (T2), HT-2 toxin (HT2), diacetoxyscirpenol, neosolaniol, and T-2 triol. Samples were also analysed for moniliformin and zearalenone by high-performance liquid chromatography (HPLC). Of the ten trichothecenes analysed from 446 harvest samples of barley, only two, diacetoxyscirpenol and neosolaniol, were not detected. The concentrations of type A trichothecenes were similar to those that occurred in wheat over the same period, whilst those of type B trichothecenes were markedly lower. Deoxynivalenol was the most frequently detected Fusarium mycotoxin, present above the limit of quantification (10 µg kg^?1) in 57% of samples, and was usually present at the highest concentration. A single sample (0.2%) exceeded the legal limit for DON in unprocessed barley over the 4-year period. Moniliformin and zearalenone were both rarely detected (2% of samples greater than 10 µg kg^?1 for both toxins) with maximum concentrations of 45 and 44 µg kg^?1, respectively. Year and region had a significant effect on DON and HT2 + T2, but there was no significant difference in the concentration of these mycotoxins between organic and conventional samples. Overall, the risk of UK barley exceeding the newly introduced legal limits for Fusarium mycotoxins in cereals intended for human consumption is very low, but the percentage of samples above these limits will fluctuate between years.     

Occurrence of bound deoxynivalenol in Fusarium head blight-infected barley (Hordeum vulgare L.) and malt as determined by solvolysis with trifluoroacetic acid

Fusarium head blight occurs worldwide in barley production, and the Fusarium mycotoxins, particularly deoxynivalenol (DON), have become a major concern for barley products, such as beer. This study investigated the content of bound DON in barley samples that were naturally infected with Fusarium head blight. Free DON was determined by gas chromatography-electron capture detection after standard acetonitrile:water extraction, while total DON was determined using treatment with trifluoroacetic acid. Bound DON is the difference between the total and free values. Bound DON was detected in approximately 40% of the samples analyzed, and represented 6 to 21% of free DON. A preliminary study indicated that both free and bound DON decrease significantly during the steeping phase of malting. These results suggest that bound DON, like the free form, may be water-soluble.     

Screening survey of deoxynivalenol in beer from the European market by an enzyme-linked immunosorbent assay

Deoxynivalenol (DON) was analysed in 313 beer samples collected from the European retail market using a commercially available immunoassay kit (enzyme-linked immunosorbent assay, ELISA). The incidence rate was about 87%, while most samples (73%) had contamination levels lower than 20 ng ml^-1. The contamination ranged between 4.0 and 56.7 ng ml^-1, with an average of 13.5 ng ml^-1. A statistically significant correlation between alcohol levels and DON contamination was found, as well as a significant difference between bottom, top and spontaneous fermenting beers. Twenty-seven beer samples were compared using a second ELISA kit and a good correlation was obtained between the two kits (r = 0.93). Although when compared with gas chromatography-mass spectrometry the ELISA tended to overestimate the results, a good correlation (r = 0.94) between the two methods was observed. Monitoring of DON in beer is important considering that DON production is dependent on the weather and that it can contribute significantly to the tolerable daily intake of DON, especially for frequent beer consumers.     

Natural occurrence of Fusarium mycotoxins of the 1990 barley crop in Korea.

During the barley harvest in June 1990, there was a great deal of rainfall and high humidity in the southern part of Korea, and natural occurrence of Fusarium mycotoxins was suspected in barley samples. The samples of undergrade barley were obtained from four provinces and analysed for the presence of deoxynivalenol (DON) and nivalenol (NIV) by gas chromatography and zearalenone (ZEN) by high performance liquid chromatography. Of 37 samples, 33, 37 and 10 were positive for DON, NIV and ZEN, respectively. The husked barley contained 29-677 ng/g for DON, 114-1546 ng/g for NIV and 183-1416 ng/g for ZEN. The naked barley contained 38-645 ng/g for DON, 85-4569 ng/g for NIV and 40-1081 ng/g for ZEN. The average concentration of NIV in naked barley was higher than that in husked barley, but the average concentration of DON in husked barley was higher than that in naked barley. The survey indicates that the 1990 barley crop in Korea was heavily contaminated with Fusarium mycotoxins.     

北京地区啤酒中赭曲霉毒素A污染调查及暴露评估

目的了解北京地区出售的啤酒中赭曲霉毒素A(ochratoxinA,OTA)的污染水平,评估本地居民对啤酒中OTA的暴露水平。方法采用免疫亲和柱-高效液相色谱-荧光检测器检测法(IAC-HPLC-FD)对啤酒样品中OTA污染水平进行测定,并参照欧盟膳食暴露评估方法进行评估。结果在检测的87份啤酒样品中,阳性样品39份,阳性率是44.83%。OTA的污染水平范围0.010～0.093ng/ml,平均污染水平为0.015ng/ml,阳性样品的平均污染水平为0.026ng/ml。居民每周从啤酒中摄入的OTA范围为0.01～0.50ng/kgBW。结论与其他国家啤酒中OTA污染水平相比,北京地区的污染水平较低。居民啤酒OTA暴露量远低于JECFA提出的100ng/kgBW的PTWI和欧盟提出的5ng/kgBW的TDI。     

The study of deoxynivalenol and its masked metabolites fate during the brewing process realised by UPLC–TOFMS method

The co-occurrence of deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON-3-Glc) has been recently reported in malt and beer. In this study, the concentration changes were monitored within the brewing process of four beer brands: light, dark tap and two lagers, produced from ground malt mixtures differing in composition, and also mycotoxins content. A simple and rapid method employing DON-dedicated immunoaffinity columns (IAC) for the selective pre-concentration, followed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UPLC–TOFMS) system for the reliable quantification at (ultra)trace levels, was validated for all experimental matrices. The results document the key role of the malt contamination nature. While in the first monitoring period a significant increase of both DON and DON-3-Glc occurred (up to 250% and 450%, respectively), fairly different trends were observed when new malts were used for identical technological processing (in some beers a decrease of DON and only a small increase of DON-3-Glc occurred). Worth noticing, that the outcome of the brewing process was surprisingly reproducible for a particular malt mixture. In the final phase, a small monitoring study comparing Czech and Austrian alcohol-free and conventional beers was carried out.     

Statistical analysis of agronomical factors and weather conditions influencing deoxynivalenol levels in oats in Scandinavia

The relationship between weather data and agronomical factors and deoxynivalenol (DON) levels in oats was examined with the aim of developing a predictive model. Data were collected from a total of 674 fields during periods of up to 10 years in Finland, Norway and Sweden, and included DON levels in the harvested oats crop, agronomical factors and weather data. The results show that there was a large regional variation in DON levels, with higher levels in one region in Norway compared with other regions in Norway, Finland and Sweden. In this region the median DON level was 1000?ng?g(-1) and the regulatory limit for human consumption (1750?ng?g(-1)) was exceeded in 28% of the samples. In other regions the median DON levels ranged from 75 to 270?ng?g(-1), and DON levels exceeded 1750?ng?g(-1) in 3-8% of the samples. Including more variables than region in a multiple regression model only increased the adjusted coefficient of determination from 0.17 to 0.24, indicating that very little of the variation in DON levels could be explained by weather data or agronomical factors. Thus, it was not possible to predict DON levels based on the variables included in this study. Further studies are needed to solve this problem. Apparently the infection and/or growth of DON producing Fusarium species are promoted in certain regions. One possibility may be to study the species distribution of fungal communities and their changes during the oats cultivation period in more detail.     

玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇双特异性抗体的制备及检测方法构建

玉米赤霉烯酮（zearalenone,ZEN）和脱氧雪腐镰刀菌烯醇（deoxynivalenol,DON）是粮食中常见的真菌毒素,长期的饮食暴露会对生物体造成严重危害。目前针对ZEN和DON同步免疫检测分别依赖各自对应的单克隆抗体,而传统单克隆抗体制备周期长、成本高。作者利用重组抗体表达技术在短时间内获得了可同时识别ZEN和DON的双特异性抗体（Bis-scFv）,并构建了基于Bis-scFv的间接竞争酶联免疫检测方法（indirect competitive enzyme-linked immunosorbent assay,IC-ELISA）,获得ZEN和DON标准曲线的半数抑制质量浓度（IC₅₀）分别为20.64 ng/mL和132.29 ng/mL;Bis-scFv具有良好的特异性,与其他真菌毒素均无显著的交叉反应。同时,将IC-ELISA方法对玉米中的ZEN和DON进行加标回收实验,其回收率为86.02%～108.14%。本研究证明了所开发的Bis-scFv未来可应用于粮食样本中ZEN与DON的同步快速检测方法的开发。     

制麦和酿造过程中脱氧雪腐镰刀菌烯醇的初步研究

2008年江苏某农场的赤霉病感染相对严重的KA-4B大麦中,脱氧雪腐镰刀茵烯醇（DON）的含量为1．91mg/kg。以此大麦为原料,实验室规模下进行制麦和酿造实验,结果表明,浸麦可以洗去大麦本身含有的绝大部分DON;而大麦内部没有被洗掉的镰孢霉属真菌孢子在发芽阶段重新萌发、生长代谢,形成并积累大量的DON;焙燥阶段不能破坏DON,成品麦芽和麦根中DON的含量分别是原大麦中DON含量的51％和89％。麦芽中的DON可以经过糖化和发酵过程流入到啤酒中,啤酒中DON的总含量是麦芽粉中DON总量的86％。而同一年份相邻农场的基本未感染赤霉病的KA-4B大麦中,DON含量低于0．1mg/kg,绿麦芽和麦根中均检测到低于0．1mg／kg的DON,而成品麦芽、麦汁和啤酒中均未检测到DON。制麦及酿造实验表明,与基本未感染赤霉病的KA-4B大麦相比,赤霉病感染程度严重的KA-4B大麦微生物污染严重,DON含量相对高,大麦品质较差,发芽率较低,麦芽浸出率低,所制得麦汁的过滤速度快,麦汁和啤酒的色度均较高。     

Survey of deoxynivalenol and its conjugates deoxynivalenol-3-glucoside and 3-acetyl-deoxynivalenol in 374 beer samples

Beer is one of the most popular beverages worldwide. Malted cereal grains are among the basic ingredients and hence mycotoxin contamination might occur. Previous studies reported the presence of the Fusarium mycotoxins deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3ADON), as well as of the masked mycotoxin deoxynivalenol-3-glucoside (D3G) in beer. In the present survey, 374?beer samples from 38?countries with a focus on Austrian (156) and German (64) beers were analysed for the presence of D3G, DON and 3ADON. Beers were assigned to the following six categories: pale (217), wheat (46), dark (47), bock (20), nonalcoholic beers (19) and shandies (25). In total, 348 and 289 beers (93 and 77%, respectively) contained D3G and DON at the levels above the limit of detection, whereas 3ADON was not detected in any of the samples. Average concentrations of all beers were 6.9?µg?L^?1 for D3G and 8.4?µg?L^?1 in the case of DON. Nonalcoholic beers and shandies showed the lowest contaminations, 1.5 and 3.2?µg?L^?1 for D3G and 2.7 and 4.4?µg?L^?1 for DON, respectively. In bock beers characterised by a higher gravity, a significant trichothecene load of 14.8?µg?L^?1 D3G and 12.4?µg?L^?1 DON was found. The highest contamination (81?µg?L^?1 D3G, 89?µg?L^?1 DON) was detected in a pale beer from Austria, underlining the importance of this study for food safety. The molar D3G to DON ratio ranged between 0.11 and 1.25 and was 0.56 on average. Concluding, the average contamination of beer is not of toxicological concern for moderate beer drinkers. However, in the case of heavy beer drinkers, beer consumption may considerably contribute to the overall intake of DON, which might even lead to exceeding the maximum tolerable limits established for this Fusarium toxin.     

Occurrence of deoxynivalenol in an elderly cohort in the UK: a biomonitoring approach

ABSTRACT

Deoxynivalenol (DON) is a Fusarium toxin, to which humans are frequently exposed via diet. Although the elderly are speculated to be sensitive to the toxic effects of DON as a result of age-related conditions, disease and altered DON metabolism, there is lack of available data on DON biomarkers in this age group. This study characterised urinary DON concentrations and its metabolites in elderly aged ≥65years (n = 20) residing in Hull, UK. Morning urinary specimens were collected over two consecutive days together with food records to assess dietary intake over a 24h-period prior to each urinary collection. Free DON (un-metabolised), total DON (sum of free DON and DON-glucuronides or DON-GlcA) and de-epoxy deoxynivalenol (DOM-1) were analysed using a validated LC-MS/MS methodology. Total DON above the limit of quantification 0.25 ng/mL was detected in the urine from 90% of elderly men and women on both days. Mean total DON concentrations on day 1 were not different from those on day 2 (elderly men, day 1: 22.2 ± 26.3 ng/mg creatinine (creat), day 2: 28.0 ± 34.4 ng/mg creat, p = 0.95; elderly women, day 1: 22.4 ± 14.6 ng/mg creat, day 2: 29.1 ± 22.8 ng/mg creat, p = 0.58). Free DON and DON-GlcA were detected in 60–70% and 90% of total urine samples, respectively. DOM-1 was absent from all samples; the LoQ for DOM-1 was 0.50 ng/mL. Estimated dietary intake of DON suggested that 10% of the elderly exceeded the maximum provisional tolerable daily intake for DON. In this single-site, UK-based cohort, elderly were frequently exposed to DON, although mean total DON concentrations were reported at moderate levels. Future larger studies are required to investigate DON exposure in elderly from different regions of the UK, but also from different counties worldwide.