冷冻-离心净化-高效液相色谱法测定植物油中黄曲霉毒素B1

通过冷冻-离心净化,建立了高效液相色谱定量测定植物油中黄曲霉毒素B1的方法,并将其用于测定市场上30批植物油产品中黄曲霉毒素B1的含量。以水/乙腈溶液为提取剂对植物油中黄曲霉毒素B1进行萃取,低温冷冻固化植物油,冷冻离心使植物油和提取液分离、净化提取液,经衍生后上液相色谱进行定量分析。优化的乙腈/水提取液配比为90:10,低温冰箱冷冻温度为-12 ℃,冷冻离心转速为12000 r/min、离心力约为1.4万 × g,以水浴加热的方式进行衍生。方法的定量检出限为0.02 μg/kg,校准曲线回归方程为y=2.321987x＋0.001377,相关系数（R2）为0.9997,在0.10～5.0 μg/L范围内线性关系良好。当样品中黄曲霉毒素B1添加量为0.5、1.0、5.0 μg/kg时,平均回收率为80.2%～93.2%,相对标准偏差为2.9%～4.7%（n=6）,均优于免疫亲和柱净化处理的结果。市售植物油产品中黄曲霉毒素B1的浓度范围为< LOD至5.72 μg/kg,均低于GB 2761—2017中对该指标的限值,花生油和玉米油中黄曲霉毒素B1均有检出,油茶籽油和核桃油中黄曲霉毒素B1均低于检出限。该方法样品前处理简便、稳定性好、检出限低,适合植物油中黄曲霉毒素B1的批量检测。     

Aflatoxin B1-degrading activity from Bacillus subtilis BCC 42005 isolated from fermented cereal products

ABSTRACT

Aflatoxin B₁ is a naturally occurring mycotoxin that is produced as secondary metabolite by Aspergillus spp., especially A. flavus and A. parasiticus. This is the most severe toxin due to its carcinogenic, mutagenic, and teratogenic properties. Hence, methods for toxin degradation have been received increasing interest from both scientific communities and industries. In this study, 32 isolates of Bacillus spp. from various fermented cereal products were screened for their aflatoxin B₁ degradation ability. The results indicated the extracellular fraction of Bacillus subtilis BCC 42005 isolated from Iru (African locust bean) potentially possessed aflatoxin B₁-degrading ability. The maximum activity of the active fraction was at 50°C and pH 8.0. The activity was stable in a wide range of pH (5.0–8.0) and temperature (25–60°C). The aflatoxin B₁-degrading mechanisms of this strain may be possibly involved by enzyme(s). This extracellular fraction was not toxic at IC₅₀ 4 mg/ml and it can be combined with water as a soaking agent for maize, which results in 54% of aflatoxin B₁ reduction after contact time 120 min. Hence, the extracellular fraction of Bacillus subtilis BCC 42005 can be further applied as an effective soaking agent in a pretreatment process with a practical and easy-to-implement condition and also probably used to reduce the aflatoxin B₁ contamination in other foods and feeds commodities.     

Aflatoxin M1 in raw milk and aflatoxin B1 in feed from household cows in Singida,Tanzania

Aflatoxin M₁ (AFM₁) contamination in raw milk from household cows fed with sunflower seedcakes or sunflower-based seedcake feeds was determined in 37 milk samples collected randomly from different locations in Singida region, Tanzania. Aflatoxin B₁ (AFB₁) contamination in sunflower-based seedcake feed was determined in 20 feed samples collected from the same household dairy farmers. The samples were analysed by RP-HPLC using fluorescent detection after immunoaffinity column clean-up. Recoveries were 88.0% and 94.5%, while the limits of detection (LOD) were 0.026 ng mL^?1 and 0.364 ng g^?1 for AFM₁ and AFB₁, respectively. Of the analysed cow’s milk samples, 83.8% (31/37) contained AFM₁, with levels ranging from LOD to 2.007 ng mL^?1, exceeding both the European Commission (EC) and Tanzania Food and Drug Authority (TFDA) limit of 0.05 ng mL^?1. Of the contaminated samples, 16.1% exceeded the Codex Alimentarius limit of 0.5 ng mL^?1. AFB₁ was present in 65% (13/20) of the feed samples with levels ranging from LOD to 20.47 ng g^?1, 61.53% exceeding the TFDA and EC maximum limits of 5 ng g^?1 for complete dairy animal feed. The observed AFM₁ and AFB₁ contamination necessitates the need to raise awareness to dairy farmers in Tanzania to safeguard the health of the end-users.     

Aflatoxin M1 in fresh milk collected from local markets of Karachi,Pakistan

During 2016–2017, 156 samples of fresh milk samples were collected from local markets of Karachi, Pakistan and analysed for aflatoxin M₁ (AFM₁) contamination using ELISA technique. AFM₁ was detected in 143 (91.7%) samples, ranged from 20 to 3090 ng L^?1 with a mean level of 346.2 ng L^?1. In 125 (80.1%) samples, the AFM₁ contamination was greater than the maximum limit (ML = 50 ng L^?1) set by EU. However, in 51 (32.7%) samples, the AFM₁ level was higher than the ML of 500 ng L^?1 as assigned by the USA. Statistical analysis showed that the AFM₁ level in milk samples from summer was significantly (p < 0.05) higher than that obtained in winter. It was concluded that the AFM₁ levels in the tested samples appear to be a serious public health problem. Therefore, immediate measures should be taken and re-evaluation done for the procedures for farming, transportation, refrigeration, and storage for the control of AFM₁ level in milk samples.     

Aflatoxin B1 and sterigmatocystin survey in beer sold in China

ABSTRACT

A total of 101 samples of beer from the Chinese market were analysed for the presence of aflatoxin B₁ (AFB₁) and sterigmatocystin (STC), using methods based on liquid chromatography–tandem mass spectrometry. The limit of quantification and the limit of detection in beer were 0.1 and 0.03 µg/kg, respectively. Recoveries of AFB₁ and STC from spiked beer samples were 97.8–103.6% and 92.7–102.1%, respectively. None of the beer purchased samples were contaminated with AFB₁ or STC.     

酶联免疫吸附法测定花生油中黄曲霉毒素B1 不确定度的评定

目的对采用ELISA方法测定花生油中黄曲霉毒素B1含量的不确定度进行分析,找出影响不确定度的因素,为评价其测定方法和检测结果的可靠性提供依据。方法根据JJF 1059.1-2012《测量不确定度评定与表示》的规定,对测定过程中标准曲线拟合、重复测量、样品的稀释、样品的称量、回收率等引入的不确定度分量进行评定。结果测定结果合成不确定度为0.45μg/kg,扩展不确定度为0.90μg/kg,花生油中黄曲霉毒素B1含量测定结果为(16.54±0.90)μg/kg(k=2)。结论测定结果不确定度主要由标准曲线拟合所引入,所建立的不确定度评定方法可用于ELISA方法测定花生油中黄曲霉毒素B1含量结果的判断。     

Aflatoxin B1 in sesame seeds and sesame products from the Greek market

Aflatoxin B₁ (AFB₁) is considered as the most potent liver carcinogen for humans. A method for determination in sesame seeds was developed. AFB₁ was extracted by methanol-water, cleaned by immunoaffinity columns and determined by high-performance liquid chromatography with fluorescence detection. The recovery factor and the limit of detection (LOD) of AFB₁ in sesame seeds were 111.5% and 0.02 ng g^?1, respectively. Thirty samples of sesame products were examined for the presence of AFB₁. After analysis, 77.6% of samples were found to be contaminated. Eight samples exceeded the European Union (EU) limit (2 µg AFB₁ kg^?1). In 15 samples, AFB₁ was below the EU limit. Seven samples remained below the LOD. The most contaminated (14.49 ng AFB₁ g^?1) sample was unpeeled packaged sesame seeds. In all samples, aflatoxigenic Aspergilli fungi as well as the risk for AFB₁ presence in sesame seed was investigated.     

Aflatoxin B1 and sterigmatocystin in wheat and wheat products from supermarkets in China

Wheat is an important cereal but it is often contaminated with mycotoxins. The natural occurrence of aflatoxin B₁ (AFB₁) and sterigmatocystin (STC) was determined in 178 food samples (32 wheat samples and 146 wheat products) purchased from Chinese supermarkets. The methodology was validated, the wheat and wheat products samples were treated with a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). From these samples 18.8% of wheat and 8.2% of cracker samples were contaminated with AFB₁. Mean levels were 0.06 µg/kg and 0.05µg/kg, respectively. There was no AFB₁ contamination in white bread or whole meal bread. Meanwhile 53.1% of wheat, 59.2% of crackers, 20.8% of white bread and 16% of whole meal bread samples were contaminated with STC. The mean levels were 0.07, 0.79, 0.12 and 0.12 µg/kg respectively. Although the levels were low, this demonstrates the need for more comprehensive surveys for these two mycotoxins in wheat and wheat products from China.     

间接ELISA检测不同贮存条件下花生中黄曲霉毒素B1

建立了一种利用间接ELISA检测花生中黄曲霉毒素B1的方法,同时考察了平衡相对湿度及贮存时间对花生中黄曲霉毒素B1(AFB1)含量的影响。结果表明:该方法的重现性好,AFB1回收率在95.20%~97.75%之间,线性范围为0.049~0.971μg/kg,最低检出限为0.029μg/kg;花生中AFB1的含量随平衡相对湿度的升高而增加,随贮存时间延长,其含量也不断增加。但平衡相对湿度低于57.6%时,即使贮存90 d,花生中的AFB1含量仍低于国家食品安全标准限量(20μg/kg)。     

Efficacy of Solis, NovasilPlus, and MTB-100 to reduce aflatoxin M1 levels in milk of early to mid lactation dairy cows fed aflatoxin B1

An experiment was conducted to determine the efficacy of 3 adsorbents, Solis (SO; Novus International Inc.), NovasilPlus (NOV; Engelhard Corp.), and MTB-100 (MTB; Alltech), in reducing aflatoxin (AF) M₁ concentrations in milk of dairy cows fed an AF-contaminated diet. Twelve early to mid lactation dairy cows averaging 163 d in milk were used in a 4 × 4 Latin square design with 3 replications. Cows were blocked by parity, body weight, and milk production and were provided ad libitum access to feed and water. Within each replicate, cows were randomly assigned to the 4 dietary treatments for 4 consecutive 7-d periods. Dietary treatments included AF [112 μg of AFB₁/kg of diet dry matter (DM)]; AF + 0.56% SO; AF + 0.56% NOV; and AF + 0.56% MTB. Milk samples were collected on d 6 and 7 of each of the experimental periods. Feed intake, milk production, milk fat percentage, milk protein percentage, and linear somatic cell scores were not affected by dietary treatments and averaged 22.20 kg/d of DM, 33.87 kg/d, 3.78%, 2.95%, and 1.60, respectively, across all treatments. Transfer rates of AF from feed to milk averaged 2.65, 1.48, 1.42, and 2.52% for cows fed AF, AF + SO, AF + NOV, and AF + MTB, respectively. Daily AFM₁ excretion in milk averaged 66, 37, 35, and 63 μg/d for cows fed AF, AF + SO, AF + NOV, and AF + MTB, respectively. The addition of SO and NOV to the AF diet resulted in a significant reduction in milk AFM₁ concentrations (SO, 45%; NOV, 48%) and AFM₁ excretion (SO, 44%; NOV, 46%). In contrast, MTB was not effective in reducing milk AFM₁ concentrations (4%), AFM₁ excretion (5%), or AF transfer from feed to milk (2.52%). Results indicated that SO and NOV at 0.56% of the diet were effective in reducing milk AFM₁ concentrations in cows consuming a total mixed ration containing 112 μg of AFB₁/kg of diet DM.     

Natural occurrence of aflatoxins (B1 and M1) in feed,plasma and raw milk of lactating dairy cows in Beja,Tunisia, using ELISA

Beja is an agricultural area in northwest Tunisia. It contributes to national needs by offering cereals and milk to the market for human and animal consumption. A small number of studies on mycotoxin occurrence in feedstuffs and raw milk from lactating dairy cows in this region are available. Therefore, 226 samples were collected from farms and local markets during November 2008 until April 2010. Samples consisted of 112 raw cow milk, 56 blood from lactating cows and 58 feed destined for dairy cows. Plasma and feed were analysed for aflatoxin B₁ (AFB₁). Milk samples were analysed for aflatoxin M₁ (AFM₁). All samples were treated using a simultaneous methanolic-aqueous extraction, followed by immunoaffinity column clean-ups and were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA). Recoveries were 80%–95% and 81%–92% for AFB₁ and AFM₁, respectively, while the limit of detection (LOD) was 0.01?µg/kg or µg/l for both mycotoxins. Results revealed the presence of AFB₁ in 84.4% of the feed samples (mean 18.7?±?1.4?µg/kg), and 39.2% of the plasma-examined samples (median 7.1?±?1.0?µg/l) were found to be contaminated at levels higher than the Tunisian and the European Union (EU) limit for dairy animals, which are 20 and 5?µg/kg in animal feed, respectively. AFM₁ was detected in 60.7% of the cow raw milk samples examined (median 13.6?±?1.4?µg/l). Contaminated levels were higher than the EU limit of 0.05?µg/l. It was concluded that more precaution should be taken on hygiene controls in order to prevent fungal contamination.     

Natural co-occurrence of ochratoxin A,ochratoxin B and aflatoxins in Sicilian red wines

The natural occurrence of ochratoxin A, ochratoxin B, aflatoxin B₁, aflatoxin B₂, aflatoxin G₁ and aflatoxin G₂ (OTA, OTB, AFB₁, AFB₂, AFG₁, AFG₂) in red wines was investigated by HPLC/FLD after immunoaffinity column clean-up in 57 market samples produced in Sicily (Italy). The results showed a very low incidence of these mycotoxins in analysed samples, confirming the high degree of quality and safety of Sicilian red wines. The results indicated 71.9% and 64.9% positive samples for OTA and OTB respectively, with an average level of 0.13 μg l^–1, well below the European maximum permitted levels (MLs). The aflatoxin most frequently detected in the samples was AFG₁, present in 57.9% of samples, while the other aflatoxins were rarely present. Recovery experiments were carried out on eight mycotoxin-free red wines spiked with OTA, OTB, AFB₁, AFB₂, AFG₁ and AFG₂ at two different levels. The limits of detection (LODs) in wines were 0.02 µg l^–1 for OTA, 0.04 µg l^–1 for OTB, 0.03 µg l^–1 for AFG₁, AFG₂ and AFB₂, and 0.05 µg l^–1 for AFB₁. A good correlation was found, with good performances in term of precision for the method.     

Effect of monogastric and ruminant gastrointestinal conditions on in vitro aflatoxin B1 adsorption ability by a montmorillonite

The main objective of this study was to evaluate the interference of environment components on the in vitro evaluation of aflatoxin B₁ adsorption capacity of sodium bentonite under simulated gastrointestinal conditions of monogastric and ruminant animals. Sodium bentonite showed a high aflatoxin B₁ affinity with all of the assays. Langmuir or sigmoid isotherms were found in different assays. Both the affinities and the surface excesses at monolayer saturation were affected by the buffer components. The specific influence of ions in each buffer solution was investigated. A significant decrease in the surface excess at monolayer saturation was observed under ionic strength control. A change in the isotherm shape from sigmoidal to Langmuir was observed with the increase in the sodium chloride concentration. This was attributed to the decrease in the importance of lateral interaction between adsorbed toxin molecules compared with surface-molecules interactions under a high salt coverage. The presence of rumen fluid components in the adsorption environment decreased the aflatoxin B₁ maximum adsorption capacity of sodium bentonite. Despite the high affinity of this adsorbent to capture aflatoxin B₁, different substances present in the environment could affect the adsorption capacity, at least at low toxin concentrations that mimic chronic exposure. The environment of the gastrointestinal tract, in either monogastric or ruminant animals, affect in vivo aflatoxin B₁ adsorption by sodium bentonite and should be taken into account when an in vitro performance evaluation is done.     

Short communication: Investigation of aflatoxin M1 levels in infant follow-on milks and infant formulas sold in the markets of Ankara,Turkey

Aflatoxins are fungal toxins known to be carcinogenic and are classified as food contaminants. This study was performed to investigate aflatoxin (AF) M₁ levels in baby foods sold in Ankara (Turkey) and to evaluate the obtained results according to the Turkish Food Codex (TFC). For this purpose, a total of 84 baby food samples (50 follow-on milks and 34 infant formulas) were obtained from different markets in Ankara and the presence of AFM₁ in the samples was analyzed by ELISA. In 32 (38.1%) of 84 infant food samples, the presence of AFM₁ was detected in concentrations ranging between 0.0055 and 0.0201 µg/kg. The mean level (±standard error) of AFM₁ was found to be 0.0089 ± 0.0006 µg/kg in positive infant follow-on milks. Aflatoxin M₁ was detected in only 1 infant formula sample (2.94%) at a concentration of 0.0061 µg/kg. The extrapolated levels of AFB₁ contamination in feedstuffs were calculated based on levels of AFM₁ in baby food samples. The data estimating AFB₁ contamination in dairy cattle feedstuff indicate that contamination may range from 0.3410 to 1.2580 µg/kg, with the mean level (±standard error) being 0.5499 ± 0.0385 µg/kg, which is lower than the level set by the TFC and European Union regulations (5 µg/kg). According to the obtained results, the levels of AFM₁ in analyzed samples were within the allowed limit (0.025 µg/kg) set in the TFC. Low levels of AFM₁ in infant follow-on milks and infant formula samples obtained during the study do not pose a health risk to infants.     

2015年山东部分地区食用植物油中黄曲霉毒素B1和玉米赤霉烯酮污染状况调查

摘 要：目的 了解山东省部分地区食用植物油中黄曲霉毒素B1和玉米赤霉烯酮的污染状况,为食品安全监督管理提供科学依据。方法 在山东省部分市/县采集食用植物油,256份8类植物油,散装油206份,定型包装油59份,其中包括花生油105份、大豆油78份、玉米胚芽油9份、香油46份、调和油10份以及其他食用油17份,酶联免疫吸附法检测AFB1和ZEN的含量,并根据食品安全国家标准食品中真菌毒素限量分析植物油中的AFB1和ZEN污染状况。结果 256份样品中,AFB1和ZEN检出率分别为44.5%、72.1%,超标率分别为7.2%、6.0%,中位数分别为0.0、4.9 μg/kg。定型包装和散装油中,AFB1超标率分别为0.0%、9.2%,差异有统计学意义（?2=4.55,P＜0.05）,中位数分别为0.6 μg/kg、0.0 μg/kg;ZEN超标率分别为10.2%、4.9%,中位数分别为5.1 μg/kg、4.8 μg/kg。各城市间AFB1总体来说差异有统计学意义（?2=32.31,P＜0.05）;ZEN在不同地区间差异无统计学意义（?2=16.06,P＞0.05）。花生油和大豆油中AFB1超标率分别为16.2%、2.6%,差异有统计学意义（?2=8.93,P＜0.05）,其他种类植物油中未发现超标;花生油、大豆油、玉米胚芽油、香油、调和油ZEN的超标率分别为1%、1.3%、100.0%、8.7%、20.0%,各类食用油中ZEN超标率差异有统计学意义（P＜0.05）。结论 山东部分地区食用植物油中AFB1和ZEN污染严重,其中以散装油污染最为严重。花生油中AFB1、玉米油中ZEN污染严重,建议相关部门加强市场散装油的监管。     

Simultaneous determination of aflatoxin B1 and M1 in milk,fresh milk and milk powder by LC-MS/MS utilising online turbulent flow chromatography

A novel, fully automated method based on dual-column switching using online turbulent flow chromatography followed by LC-MS/MS was developed for the determination of aflatoxin B₁ and M₁ in milk, fresh milk and milk powder samples. After ultrasound-assisted extraction, samples were directly injected into the chromatographic system and the analytes were concentrated on the clean-up loading column. Through purge switch, analytes were transferred to the analytical column for subsequent detection by mass spectrometry. Different types of TurboFlow^TM columns, transfer flow rates and transfer times were optimised. Method limits of detection obtained for AFB₁ and AFM₁ were 0.05 μg kg^–1, and limits of quantification were 0.1 μg kg^–1. Recoveries of aflatoxin B₁ and M₁ were in range of 81.1–102.1% for all samples. Matrix effects of aflatoxin B₁ and M₁ were in range of 63.1–94.3%. The developed method was successfully used for the analysis of aflatoxin B₁ and M₁ in real samples.     

纳米免疫传感器法快速测定粮油食品中的黄曲霉毒素B1

摘要：本文以改性壳聚糖为固定化材料,包埋固定纳米金胶微粒及黄曲霉毒素B1抗体制备信号放大型纳米免疫传感器,建立了免疫传感器测定黄曲霉毒素B1的方法;优化了纳米免疫传感器的制备条件及检测参数;基于AFB1抗体与抗原之间的特异性免疫反应,以K3[Fe(CN)6]为探针,利用循环伏安法和差分脉冲伏安法研究了其免疫反应对传感器响应电流的影响,结果表明免疫响应电流与底液中AFB1的浓度在0.1～1.1 ng mL-1范围内成线性关系,其校正曲线方程为IP =-4.9274x +15.108（R 2= 0.9912）,其最低检测限为0.05 ng mL-1（S/N=3）;该免疫传感器的稳定性和重现性较好。利用该法对花生油、玉米油等实际样品中的AFB1进行了检测,其回收率为在87.8～98.2%,检测精确度优于ELISA试剂盒法,用于粮油食品中黄曲霉毒素的快速检测是可行的。     

Activated carbons as potentially useful non-nutritive additives to prevent the effect of fumonisin B1 on sodium bentonite activity against chronic aflatoxicosis

Aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) are mycotoxins that often co-occur in feedstuffs. The ingestion of AFB₁ causes aflatoxicosis in humans and animals. Sodium bentonite (NaB), a cheap non-nutritive unselective sequestering agent incorporated in animal diets, can effectively prevent aflatoxicosis. Fumonisins are responsible for equine leukoencephalomalacia and porcine pulmonary oedema, and often have subclinical toxic effects in poultries. Fumonisin B₁ and aflatoxin B₁ are both strongly adsorbed in vitro on sodium bentonite. Co-adsorption studies, carried out with a weight ratio of FB₁ to AFB₁ that mimics the natural occurrence (200:1), showed that FB₁ greatly decreases the in vitro ability of NaB to adsorb AFB₁. The ability of two activated carbons to adsorb FB₁ was also investigated. Both carbons showed high affinity for FB₁. A complex behaviour of the FB₁ adsorption isotherms with pH was observed. In vitro results suggest that under natural contamination levels of AFB₁ and FB₁, a mixture of activated carbon and sodium bentonite might be potentially useful for prevention of sub-acute aflatoxicosis.     

乳粉中维生素B1和B2含量测定的能力验证分析

目的为加强我国检测机构对乳粉市场的监管,中国食品药品检定研究院(National Institute for Food and Drug Control,NIFDC)组织实施了乳粉中维生素B_1和B_2的含量测定的能力验证。方法制备了分割水平对的样品,采用单因素方差分析和t检验对样品均匀性和稳定性进行分析。对能力验证结果进行稳健统计分析,通过Z比分数评价实验室检测能力。结果样品通过均匀性和稳定性检验要求,满足能力验证计划要求。维生素B_1项目中,参加20个实验室验证,满意结果数为14,满意率为70.0%;维生素B_2项目中,参加22个实验室验证,满意结果数为15,满意率为77.2%;2项检测结果均满意的实验室有12个,满意率为60%。结论绝大多数实验室对乳粉中维生素B_1和B_2的测定处于良好水平,但未达到优良水平,仍需对检测方法进行统一认识,减少系统误差,加强各实验室内部的质量控制。     

微生物法针对毒性位点降解黄曲霉毒素B1的研究进展

文章主要探究了黄曲霉毒素B₁(AFB₁)的毒性位点及毒性机理,并对比了微生物法破坏AFB₁毒性位点从而降解AFB₁的优势。重点综述了微生物法中的脱毒酶法对AFB₁的降解机制。同时,对脱毒酶法联合其他方法的复合应用进行展望。