Formation of a derivative of aflatoxin B1 on a sephadex column with a 1% aqueous solution of methanol

Summary A partial separation of aflatoxins B₁ and B₂ on a Sephadex column with a 1% aqueous solution of methanol as mobile phase was achieved. The chromatographic system is, however, not neutral for aflatoxin B₁. During elution a derivative-probably aflatoxin B₁. hemiacetalis produced. The derivative forms blue fluorescent spots on Adsorbosil-1 plates with R_f 0.15 when developed in chloroform/acetone (90 + 10).

---

Bildung von Umwandlungsprodukten aus Aflatoxin B₁ während der Chromatographie an Sephadex mit 1%iger Lösung von Methanol in Wasser

Zusammenfassung Man erreichte eine teilweise Trennung der Aflatoxine B₁ und B₂ auf der Sephadex Säule mit l % iger Lösung von Methanol in Wasser. Dieses chromatographische System ist jedoch gegen Aflatoxine B₁ nicht neutral. Während der Elution bildet sich ein Derivat, wahrscheinlich Hemiacetal des Aflatoxin B₁. Das Derivat gibt blaue Fluorescenzflecken auf Adsorbosil-1 Platten wenn mit Chloroform/Aceton (90 + 10) entwickelt.

     

基于壳聚糖微球的黄曲霉毒素B1免疫亲和柱开发制备

目的 基于壳聚糖微球制备一种用于黄曲霉毒素B1检测前处理的免疫亲和柱。方法 通过优化化学交联法的反应条件,包括壳聚糖分子量、氨基和醛基摩尔比、壳聚糖浓度和水相油相比,制备粒径大小合适且均一的壳聚糖微球,并以此为载体偶联黄曲霉毒素B1的单克隆抗体制备免疫亲和柱。对亲和柱性能进行分析,使用阳性实际样品对亲和柱使用效果进行验证。结果 确定了壳聚糖微球的最佳制备条件。在此条件下制备的免疫亲和柱非特异性吸附率为5.26%,对单克隆抗体偶联率为90.90%,柱容量为2.69 μg AFB₁/mL壳聚糖微球,检测回收率为92.1%~98.6%,相对标准偏差为0.61%~2.53%。用于实际样品检测回收率为87.90%~96.37%。 结论 本研究建立的免疫亲和柱制备过程简单,成本低,为黄曲霉毒素B1痕量检测前处理提供一种新的方案。     

Nisin extraction capacity of aqueous ethanol and methanol from a 2.5% preparation

Nisin is a widely used antimicrobial preservative whose efficacy is compromised by interaction with food components when applied to food matrices hence, encapsulation may overcome this problem. The objective of this research was to study parameters for concentrating nisin from a 2.5% preparation using 10–100% aqueous ethanol and methanol at 1–15 mg solids/mL solvent (MSMS). The extraction yield was the highest at an intermediate alcohol concentration (80% methanol or 50% ethanol) corresponding to intermediate polarity, while a poorer extraction was observed at higher MSMS. The higher alcohol concentration also enabled improved removal of impurities such as protein. The highest purification factor (PF) was observed at 1 MSMS in 90% methanol (PF = 5.3, 91.0% yield) or 2 MSMS in 70% ethanol (PF = 5.5, 84.7% yield). The recommended conditions (2 MSMS in 70% ethanol) were further used to prepare a powdered product 4.4 times more concentrated by spray drying. The concentrated product may be used for encapsulation of the antimicrobial in capsules or films/coatings.     

免疫亲和柱净化-光化学衍生高效液相色谱荧光法测定植物油中黄曲霉毒素B1的含量

建立了免疫亲和柱净化-光化学衍生高效液相色谱荧光法测定植物油中黄曲霉毒素B1含量的方法,并对样品的测定条件进行了优化。结果表明:植物油的称样量缩减为5.00 g,黄曲霉毒素B1测定结果与GB/T 18979—2003测定结果基本吻合;工作曲线在1~40 ng/mL质量浓度范围内具有良好的线性关系,相关系数为0.999 9,方法检出限为0.3μg/kg;在空白样品中添加低、中、高3个不同添加量水平的黄曲霉毒素B1标准品,其回收率在90.5%~99.8%之间,相对标准偏差在6.3%~9.8%之间。采用光化学衍生,操作简单,无需衍生剂,避免了柱前衍生和柱后衍生受衍生剂浓度、温度、反应时间的影响,所建立的方法适用于植物油中黄曲霉毒素B1含量的测定。     

Effect of dietary acids on the formation of aflatoxin B2a as a means to detoxify aflatoxin B1

Aflatoxin B₁ (AFB₁) is a class 1 carcinogen and a common food contaminant worldwide with widely uncontrolled human exposure. The ability of organic acids to transform AFB₁ into a known detoxified form, aflatoxin B_2a (AFB_2a), was investigated using high performance liquid chromatography-electrospray ionisation-time of flight mass spectrometry (HPLC/ESI/TOF/MS). The identity of the transformation product was confirmed by accurate mass measurement, chromatographic separation from other aflatoxins, H¹-nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. Of the weak acids tested, citric acid was found to be the most effective for AFB_2a formation. At room temperature, 1 M citric acid was able to convert > 97% of AFB₁ to AFB_2a over 96 h of treatment. Up to 98% transformation was achieved by boiling AFB₁ in the presence of citric acid for 20 min. AFB₁ hydration after ingestion was explored by spiking AFB₁ into simulated gastric fluid containing citric acid. Under these conditions, > 71% of AFB₁ was hydrated to AFB_2a and did not show any reversion to the parent compound after being transferred to a neutral solution. These results provide a basis for a practical and effective method for detoxification of AFB₁ in contaminated foods.     

碱液处理花生脱除花生油中黄曲霉毒素B1的研究

用碱液处理花生,检测压榨花生油中的黄曲霉毒素B_1(AFB_1)的含量及花生油质量指标。研究表明:碱液处理花生对脱除花生油中黄曲霉毒素B_1具有良好的效果。采用碱液pH 13. 40、碱液加入量10%的条件处理四级花生15 min,压榨花生油中AFB_1脱除率达93. 3%,采用p H 13. 44、碱液加入量7. 5%的条件处理三级花生25 min,压榨花生油中AFB_1脱除率达98. 0%。碱液处理对花生油的酸价有一定影响但在标准规定范围内,对过氧化值、碘值及脂肪酸组成无确定影响关系。     

应用抗黄曲霉毒素单链抗体检测酱油中黄曲霉毒素B1

利用本实验室制备的抗黄曲霉毒素B1的单链抗体(ScFv),通过棋盘实验确定了抗原抗体的最适工作浓度,在此之上根据间接竞争酶联免疫法(ELISA)绘制标准曲线,检测酱油中AFB1的含量;通过改变样品的盐浓度及pH来确定其对ELISA检测结果的影响。研究结果表明,利用ScFv检测黄曲霉毒素的最小检测值为0.10ng/mL,平均加标回收率在84%～109%之间,本文建立的ELISA方法在pH5～8,盐浓度小于10%时较稳定。本文建立的利用抗AFB1的ScFv检测黄曲霉毒素含量的方法方便快捷,稳定性较好,并且成本较低,适合于食品中黄曲霉毒素的检测。     

Excretion pattern of aflatoxin M1 in milk of goats fed a single dose of aflatoxin B1

The feedstuffs used in dairy animals must be able to give consumers confidence about the wholesomeness of milk with regard to aflatoxin contamination. The aim of this study was to determine the excretion patterns of aflatoxin M(1) (AFM1) in the milk of dairy goats fed a single dose of pure aflatoxin B(1) (AFB1), which can occasionally occur if feeds are infected by hot-spot growth of molds that produce aflatoxins. Five dairy goats in midlactation were administered 0.8 mg of AFB1 orally. Individual milk samples were collected for 84 h after AFB1 dosage. Aflatoxin M(1) was found in milk in the highest concentration. In all goats, AFM1 was not detected in milk before AFB1 administration, but was detected in the first milking following AFB1 administration. The excretion pattern of AFM1 concentration in milk was very similar in all goats even if the values of the concentration differed between animals. The peak values for AFM1 concentration in milk was observed in milk collected during the milking at 3 and 6h. After the peak, the AFM1 in milk disappeared with a trend that fitted well a monoexponential decreasing function, and the toxin was not detected after 84 h. Only about 0.17% of the amount of AFB1 administered was detected as AFM1 in milk, and about 50% of this was excreted in the first liter of milk yielded after AFB1 intake. Correct procedures to prevent growth of molds, and consequent AFB1 contamination, on the feedstuffs for lactating goats represent the key to providing consumers a guarantee that milk is not contaminated by AFM1.     

Excretion of aflatoxin M1 in milk of dairy ewes treated with different doses of aflatoxin B1

Two experiments were conducted to study the amount of aflatoxin M1 (AFM1) in milk in response to feeding aflatoxin B1 (AFB1). In experiment 1, four dairy ewes in early lactation received a single dose of pure AFB1 (2 mg). Individual milk samples were collected during the following 5 d to measure AFM1 concentration. The average excretion of AFM1 in milk followed an exponential decreasing pattern, with two intermediate peaks at 24 and 48 h. No AFM1 was detected in milk at 96 h after dosing. The mean rate of transfer of AFB1 into AFM1 in milk was 0.032%, with a high individual variability (SD = 0.017%). In experiment 2, 16 dairy ewes in midlactation were divided into four groups that received different daily doses of AFB1 (0, 32, 64, and 128 microgram for control and groups T1, T2, and T3, respectively) for 14 d. Pure AFB1 was administered to each animal divided in two daily doses. Individual milk samples were collected at 12, 24, 36, 48, 72, 96, 144, 216, and 312 h after the first AFB1 administration, during the intoxication period, and every 24 h for 7 d after the withdrawal of AFB1. AFM1 was detected in the milk of all animals of the treated groups at 12 h after the administration of AFB1. In all treated groups, milk AFM1 concentration increased from 12 to 144 h after the beginning of administration. It then decreased, reaching a stable concentration at 216 and 312 h after the first administration. No AFM1 was detected in milk 3 d after the last administration of AFB1. Milk AFM1 concentration measured at steady-state condition was significantly affected by the AFB1 dose (0.031, 0.095, and 0.166 in T1, T2, and T3 groups, respectively), with a linear relationship between AFB1 dose and milk AFM1 concentration (R2 = 77.2%). The carryover (AFM1/AFB1 ratio) was not significantly affected by treatment, and its mean value was 0.112% (SE = 0.011). The carryover was lower than that reported for dairy cattle and goats, suggesting a better ability of sheep to degrade AFB1.     

莱鲍迪苷A在甲醇-水体系中溶解度与超溶解度的测定

测定了莱鲍迪苷A在甲醇溶液中的溶解度和超溶解度曲线,并用数学模型进行了拟合,同时还测定了温度、降温速率、搅拌速率对甲醇溶液中莱鲍迪苷A介稳区宽度的影响。结果表明,莱鲍迪苷A在甲醇溶液中的溶解度随着温度的升高而增加,而介稳区宽度随着温度的升高而减小,随着降温速率的增加而增大,随着搅拌速率的增加先减小后增大。     

Determination of aflatoxin B1 levels in peanut butter using an immunoaffinity column clean-up procedure: inter-laboratory study

Ten United Kingdom laboratories participated in an evaluation of an immunoaffinity column sample preparation procedure used to prepare aflatoxin B1 containing extracts obtained from peanut butters contaminated by aflatoxins. Each laboratory was sent seven randomly numbered samples of roasted peanut butter which included two sets of undisclosed triplicates. These two peanut butters were naturally contaminated with aflatoxin B1 at levels of about 12 and 35 micrograms/kg. The other sample was a nominal blank peanut butter containing approximately 2 micrograms/kg of aflatoxin B1 which was also employed by participants for recovery experiments. Participating laboratories were instructed to follow a protocol regarding the use of the immunoaffinity columns for extract preparation, but were allowed a free choice of instrumental technique for quantification of aflatoxin levels. Mean recovery for spikes was 72%. Coefficients of variation for the results from the 10 participants for the two contaminated roasted peanut butters were, respectively, 45% (on a mean of 13.6 micrograms/kg) and 36% (on a mean of 37.2 micrograms/kg).     

黄曲霉毒素B1的生物脱毒研究进展

简要介绍了黄曲霉毒素的分子结构、理化性质、对人体健康的危害及黄曲霉毒素B1在食品中的限量标准。重点阐述了近几年黄曲霉毒素B1生物脱毒方面的进展,其中微生物主要通过吸附和降解两方面去除黄曲霉毒素B1。     

常压等离子体降解黄曲霉毒素B1的效果研究

采用常压等离子体对乙腈中黄曲霉毒素B_1(AFB_1)进行降解。利用单因素实验,考察了放电间距、处理电压、放电时间以及AFB_1初始浓度对AFB_1降解率的影响,在此基础上进行了BoxBehnken的实验设计,选取AFB_1降解率作为响应值,优化了AFB_1的降解条件。结果表明:各因素对AFB_1降解率的影响大小依次为处理电压放电时间AFB_1初始浓度。常压等离子降解AFB_1的最佳工艺条件为处理电压170 V、放电时间236 s、AFB_1初始浓度5 mg/L、放电间距2 cm。AFB_1的降解率高达92.45%,与预测值93.94%相接近,偏差为1.49%。     

Binding of aflatoxin B1 by probiotic bacteria

The ability of six probiotic bacteria to bind a common food carcinogen, aflatoxin B₁, was assessed. The studied strains included Lactobacillus strains and one Bifidobacterium strain. The strains were incubated in vitro with alfatoxin B₁ and the toxin residue in the supernatant was measured using high‐performance liquid chromatography. The aflatoxin‐binding capacity of the strains was found to range from 5.8 to 31.3%. The results further support the observation that a number of probiotic bacteria are able to bind specific dietary contaminants. Although the extent of binding varies depending on the bacterial strain used, the data may explain some of the antimutagenic and anticarcinogenic effects of probiotic micro‐organisms. © 2000 Society of Chemical Industry     

黄曲霉毒素新型抗体制备研究进展

免疫学检测方法具有快捷、灵敏、特异性高的特点,在毒素的定量和定性方面已获得了快速的发展,成为毒素检测方法的研究热点。而高质量抗体的制备是建立特异性强、灵敏度高的免疫分析方法关键。目前主流研究和应用的抗体是单克隆抗体,其性质稳定,特异性强,灵敏度高。随着抗体技术的发展,重组抗体在免疫检测领域也逐渐应用。与多克隆抗体、单克隆抗体相比较而言,重组抗体具有独特的优势,可以在原核表达体系里短时间内大量生产且生产费用低廉,对黄曲霉毒素的低成本、大规模检测有重要的应用价值。本文重点阐述和分析了黄曲霉毒素单克隆抗体、重组抗体制备过程中存在的影响因素及问题,并对未来黄曲霉毒素抗体的发展前景进行了展望。     

茶叶中黄曲霉毒素B1的检测方法研究

目的针对茶叶中黄曲霉毒素B1的检测,对比胶体金免疫层析法、高效液相色谱、酶联免疫法的差异。方法以红茶、绿茶、花茶为基质,进行方法验证。并选取具有代表性的红茶、绿茶、乌龙茶、及花茶、萃取茶、药用保健茶分别用不同的方法检测。结果改良后的液相法和胶体金免疫层析法准确度和精密度较高,酶联免疫法存在假阳性。随机检测11种茶叶仅有一种检出黄曲霉毒素B1。     

Maize populations with resistance to field contamination by aflatoxin B1

Maize (Zea mays L) germplasm is needed with resistance to infection by Aspergillus flavus and/or subsequent contamination by aflatoxin B₁ (AFB₁). A select group of maize populations were evaluated for their resistance to AFB₁ contamination at three locations. Four populations (Ibadan B and three others derived from crosses between Corn Belt inbreds and diploid perennial teosinte. Zea diploperennis) were compared with a susceptible hybrid check in a randomised complete block experiment with 10 replicates in Georgia, Missouri and South Carolina for two years. Ears were not inoculated, and naturally occurring concentrations of AFB₁ in harvested grain were analysed for population differences. Ibadan B and Mo2 W × Z diploperennis had significantly lower average amounts of AFB₁, 19 μg kg^?1 and 18 μg kg^?1, respectively, than other test entries. Backcrossing to susceptible Corn Belt inbreds produced populations as susceptible as the check when resistance was measured as the concentrations of AFB₁ in the grain. The consistency and significance of low AFB₁ concentrations for Ibadan B and Mo20W × Z diploperennis suggest that these may be useful sources of resistance.     

全自动免疫磁珠法与免疫亲和柱法测定食用油中黄曲霉毒素B1的对比研究

采用全自动免疫磁珠净化与免疫亲和柱净化的前处理方法,结合超高效液相色谱法对不同食用油中黄曲霉毒素B₁进行定量检测,并对两种前处理方法结果准确性、加标回收率、检测时间进行了对比。结果表明：全自动免疫磁珠净化与免疫亲和柱净化测定植物油标准物质,结果分别为15.59μg/kg和14.64μg/kg,均在标准值范围内;对不同种类食用油添加不同量的黄曲霉毒素B1标准物质,加标回收率均在88%～110%之间,相对标准偏差均在5%以内。采用Bland-Altman法对全自动免疫磁珠净化法和免疫亲和柱净化法进行差异分析,结果显示这两种净化方法的差异在可接受范围内。两种方法在净化和富集食用油中黄曲霉毒素B₁均可满足实验要求,可以互相代替使用。通过检测时间对比,全自动免疫磁珠净化搭配使用真菌毒素全自动净化仪前处理方法可同时处理多个样品,平均一个样品净化时间小于3 min,处理时间短、实验效率更高。     

Effect of beta-naphthoflavone on calf liver metabolism of aflatoxin B1

Eight male Holstein calves were raised to 6 wk of age on whole milk at 8% of body weight and calf starter ration (15% crude protein) free choice. For each of 2 days prior to sacrifice, four of the calves were selected at random and administered the mixed function oxidase inducer beta-napthoflavone by bolus in starch carrier at .005% of metabolic body weight. After sacrifice, 10,000 X g postmitochondrial supernatant preparations were made from the excised livers and incubated with a 50-micrograms challenge of aflatoxin B1. Although there was no significant difference in total percent of the challenge metabolized, there was a large increase in aflatoxin M1 production by liver preparations from calves administered the flavone inducer. If the flavone family of inducers could alter the metabolic profile for aflatoxin in the adult lactating bovine, aflatoxin M1 excretion rates could be increased and pathways for production of mutagenic metabolites could be enhanced.