Aflatoxin M1 Contamination of Milk and Its Products in Bomet County, Kenya

Aflatoxin M1 (AFM1) is a major carcinogenic compound that may be found in milk and dairy products resulting from ingestion of aflatoxin B1 by dairy animals. The study aimed at determining the level of aflatoxin M1 in milk and milk products from Bomet County. A total of 185 samples (150 raw milk and 35 processed milk and milk products) were randomly collected from milk collection sites and randomly selected milk kiosks respectively. The AFM1 was analyzed using a commercial ELISA kit (Ridascreen, aflatoxin M1 R-Biopharm, Product code, R5812, Darmstadt, Germany). Out of 185 samples investigated, 156 samples were positive for AFM1, an overall contamination rate of 84.32%. The samples with levels higher than the tolerance limit of 0.05 μg/l recommended by Food and Agriculture Organization (FAO) and World Health Organization (WHO) limits were 43.8% mainly contributed by the raw milk compared to processed milk (52.0% versus 8.6%). Processed milk had insignificant level of contamination with aflatoxin M1 (Median 0.00 (IQR: 0.00, 0.00 μg/l) with a minimum of 0.00 μg/l and a maximum of 0.69 μg/l. Raw milk showed significant contamination, median 0.09 (IQR: 0.00, 0.50) μg/l with a minimum of 0.00 μg/l and a maximum of 2.93 μg/l. Although there was no significant differences in AFM1 levels with study sites (P = 0.217);the median levels of aflatoxin M1 was high in sites 1, 3, and 7. The sites that had median aflatoxin M1 levels below the WHO/FAO acceptable limits of 0.05 μg/l were sites 2, 4 and 6. Due to high incidence of AFM1 contamination of milk and milk samples in Bomet County, there is need for regular monitoring and regulation of AFM1 contamination in milk and its products in the County.

Reducing Efficiencies of the Commonly Used Heat Treatment Methods and Fermentation Processes on Aflatoxin M1 in Naturally Contaminated Fresh Cow Milk

The reducing efficiencies of the commonly used heat treatment methods and fermentation processes on aflatoxin M1 (AFM1) in Nigeria were investigated. Seventy samples of fresh cow milk from both conventional and traditional dairy cattle herds were collected and analyzed for the determination of AFM1 using Cobra-cell incorporated high performance liquid chromatography. Of these analyzed samples, 56 (80.0%) tested positive for AFM1 out of which 3 milk samples with high AFM1 concentrations were selectively pooled and subjected to varied conditions of heat treatments and fermentation processes using both indigenized and exotic strains of lactic acid bacteria (Lactobacillus bulgaricus + Streptococcus thermophilus and L. rhamnosus and L. plantarum) as starter cultures respectively. Both processes used either singly or combined, demonstrated high degrees of reducing effects on AFM1 levels. Sterilization of the milk at 121?C and 80?C under the same condition of time (15 - 20) min showed significant reduction of up to 58.8% (p 0.05) in the level of AFM1 when compared with the initial mean AFM1 concentration of the untreated fresh milk. The situation was however different around the boiling temperature of 100?C at which point the level of AFM1 reduction was found to be inconsistent. The indigenized combined strains showed some slight margins of AFM1 reduction in the proportions of (20.5, 30.8 and 43.9)% over and above that of the exotic strains (17.4, 30.0 and 41.1)% in 12 h, 48 h and 72 h of fermentation respectively. Generally, fermentation alone showed lower reduction of AFM1 in milk from 24.5% to 43.9% compared with the reducing activities of (35.4 to 58.8)% when heat-treated milk samples were subsequently subjected to varied fermentation conditions.

抗黄曲霉毒素M1纳米抗体的筛选与初步鉴定

黄曲霉毒素M1(aflatoxin M1,AFM1)对人类和动物具有致癌性、诱变性、致畸性等,广泛存在于哺乳动物乳汁及其乳制品中。因此,制备特异性强、灵敏度高的抗体来检测AFM1至关重要。该研究以AFM1-BSA为抗原免疫双峰驼,采血分离淋巴细胞,提取总RNA反转录为cDNA,通过巢式PCR获得重链可变区(variable domain of heavy chain,VHH),与pComb3XSS载体连接电击转化至Escherichia coli TG1,构建AFM1特异性噬菌体展示文库。以AFM1-OVA为靶向抗原,使用4轮酸洗脱和竞争洗脱相结合的策略对文库进行淘选,并对抗AFM1纳米抗体进行初步鉴定,最后预测了精准的三维模型结构。结果显示,抗AFM1纳米抗体文库库容量为2.27×10^(9)CFU,多样性丰富,插入率为100%。经淘选共获得6条独特序列的纳米抗体。其中VHH-M4和VHH-M6具有较高的灵敏度,初步测定半抑制浓度分别为0.415 ng/mL和1.877 ng/mL,线性范围分别为0.141~1.652 ng/mL和0.800~3.811 ng/mL。一级和二级结构结果显示VHH-M4和VHH-M6均为亲水性蛋白,框架区(framework region,FR)和互补决定区(complementarity determining region,CDR)区符合典型纳米抗体的结构特征。该研究为AFM1免疫学检测提供核心元件,并为探究AFM1与VHH的分子结合机制奠定基础。

基于纳米抗体建立icELISA检测乳制品中黄曲霉毒素M1

纳米抗体(nanobody,Nb)具有稳定性高、特异性强、易于表达等优势,已经成为小分子免疫分析的重要工具。为了监测乳制品中黄曲霉毒素M1(aflatoxin M1,AFM1)的污染情况,以先前从双峰驼AFM1免疫文库中淘选得到的抗AFM1型纳米抗体M6为研究对象,评估其理化性质并建立间接竞争酶联免疫吸附测定法(indirect competitive enzyme-linked immunosorbent assay,icELISA)。优化反应体系中封闭方法、甲醇溶液、pH值和离子强度等参数后,建立竞争抑制标准曲线,并进行方法学验证特异性和准确性。结果表明,Nb-M6具有良好的亲和力和热稳定性;Nb-icELISA的检测限(limit of detection,LOD)为0.111 ng/mL,半数抑制浓度(half maximal inhibitory concentration,IC_(50))为1.498 ng/mL,乳制品加标回收率为89.8%~104.1%,与高效液相色谱法的检测结果趋于一致。该方法操作简单、便捷,可应用于乳制品中AFM1的初步批量筛查。

酶联免疫吸附和胶体金技术对黄曲霉毒素M1检测的结合验证

[目的]为准确完成生牛乳中黄曲霉毒素M1检测。[方法]对比分析酶标仪、胶体金读数仪检测生牛乳中黄曲霉毒素M1的处理过程和检测数据。[结果]发现2种方法检验操作过程各有优劣,检测结果能满足不同需求。酶标仪可用于半定量检测,并进行精准检测和半定量复核。胶体金读数仪适合快速定性筛查。[结论]2种方法可配合使用,相互补充,灵活运用设备优势,高效完成检测需求。

快速检测乳及乳制品中黄曲霉毒素M1的方法研究

黄曲霉毒素M1(AFM1)作为黄曲霉毒素的一种代谢产物,主要存在于生鲜乳及各种乳制品中。它属于二氢呋喃杂萘邻酮衍生物,呈无色结晶性粉末状,有绿蓝色荧光,物理化学性质稳定,具有很强的致癌性,且当乳品经过高温消毒后仍无法完全去除该毒素[1]。建立乳制品中黄曲霉毒素M1的高灵敏度、快速检测技术,对保障乳制品质量安全及消费者健康具有重大意义。

An overview of aflatoxin B1 biotransformation and aflatoxin M1 secretion in lactating dairy cows

Milk is considered a perfect natural food for humans and animals.However,aflatoxin B1(AFB1)contaminating the feeds fed to lactating dairy cows can introduce aflatoxin M1(AFM1),the main toxic metabolite of aflatoxins into the milk,consequently posing a risk to human health.As a result of AFM1 monitoring in raw milk worldwide,it is evident that high AFM1 concentrations exist in raw milk in many countries.Thus,the incidence of AFM1 in milk from dairy cows should not be underestimated.To further optimize the intervention strategies,it is necessary to better understand the metabolism of AFB1 and its biotransformation into AFM1 and the specific secretion pathways in lactating dairy cows.The meta-bolism of AFB1 and its biotransformation into AFM1 in lactating dairy cows are drawn in this review.Furthermore,recent data provide evidence that in the mammary tissue of lactating dairy cows,aflatoxins significantly increase the activity of a protein,ATP-binding cassette super-family G member 2(ABCG2),an efflux transporter known to facilitate the excretion of various xenobiotics and veterinary drugs into milk.Further research should focus on identifying and understanding the factors that affect the expression of ABCG2 in the mammary gland of cows.

Removal of aflatoxin M1 from milk and aqueous medium by indigenously isolated strains of W.confusa H1 and L.plantarum S2

Employing LAB and/or their metabolites to reduce aflatoxins from food products is one of the fascinating areas of research.Therefore,in the present work an attempt was made to study the effect of isolated lactic acid bacteria(LAB)on the reduction of aflatoxin M1(AFM1)from phosphate buffer saline(PBS)and reconstituted skim milk(RSM).Out of 68 isolates,nine strains of LAB isolated from food products were examined to bind and reduce AFM1.The isolates were incubated with AFM1 at different time and temperature(24 h,48 h,and 72 h at 37℃ and 4℃)and later the residual toxin in the supernatant was determined.The preliminary results through thin layer chromatography(TLC)revealed that all isolates were able to bind AFM1 in PBS.However,based on the intensity of spots observed in TLC sheets,it was indicated that two isolates,H1 and S2 genotypically identified as W.confusa and L.plantarum shown better AFM1 binding ability as compared to other isolates.Further,the heat treated cells of both the selected strains did not show significant difference to AFM1 binding ability as compared to live cells.Results of high performance liquid chromatography(HPLC)observed that W.confusa H1 and L.plantarum had shown 78% and 72%AFM1 binding in PBS at 37℃ after 72 h.There was a significant difference(P<0.05)in AFM1 binding ability of L.plantarum S2 at 37℃&4℃,but such difference was not observed for W.confusa H1.Moreover,these isolates also observed reduction of naturally present AFM1 in 10%RSM.This is the first study reporting the AFM1 binding capability of the genus Weissella(strain W.confusa H1)and Enterococcus durans.These results showed that indigenous strains of LAB are able to bind AFM1 effectively.

乳及乳制品中黄曲霉毒素M1研究进展

随着我国经济的发展与人们生活水平的提高,具有丰富营养价值的乳及乳制品逐渐成为人们日常生活的必需消费品。与此同时,人们对乳及乳制品的期待不再局限于量的提高,而是对其质量和安全提出了更高的要求。由于2011年发生的牛奶中黄曲霉毒素M_1(AFM_1)含量超标事件,以及相比于成人,婴幼儿更易受到牛奶中AFM_1的损害,人们开始更加重视乳及乳制品中存在的AFM_1污染问题。本文在国内外已有文献报道基础上,对乳及乳制品中AFM_1的来源与生物学性质、污染与限量标准、检测与防控技术进行综述。

抗黄曲霉毒素M1单克隆抗体的制备及定量ELISA方法的建立

采用黄曲霉毒素M1与牛血清白蛋白偶联物免疫8周龄BALB/c小鼠,4次免疫后,使小鼠产生免疫应答,取其脾细胞与Sp2/0骨髓瘤细胞融合、筛选,最终获得1株能稳定分泌抗黄曲霉毒素M1的杂交瘤细胞株系。其染色体平均计数为90±10对,间接ELISA检测该株系培养上清的效价为1∶6400,纯化腹水效价为5000×27,与黄曲霉毒素M1及其结构类似物黄曲霉毒素B1和脱氧雪腐镰刀菌烯醇的反应率分别为100%、7%、0%。经传30代后,抗体效价稳定。并在此基础上建立了间接竞争ELISA检测方法,该方法的最低检出浓度是0.08ng/mL,校正曲线的线性范围为0.08ng/mL～5ng/mL。该方法的加标回收率为80.0%～128.3%。

降解黄曲霉毒素M1菌株筛选方法的建立与菌株鉴定

为实现对黄曲霉毒素M1(Aflatoxin M1,AFM1)的生物降解,建立从环境中筛选AFM1降解菌株的方法并得到降解菌株。结果表明:对采自农田、粮库、牛场的300个土壤或粪便样品,初筛选出能在以香豆素为唯一碳源的培养基上生长的菌株37株;用液相色谱法测定降解率进行复筛,得到3株降解率在50%以上的菌株;进而对复筛菌株进行生长条件和耐受性实验,筛选出降解率为78.6%,生长速度快、长势好、稳定期长,且降解过程中热稳定性好的菌株102807。经过菌落细胞形态学、生理生化特征及16S r DNA序列系统发育分析,鉴定为纤维纤维微菌(Cellulosimicrobium cellulans)。首次发现了纤维纤维微菌对AFM1的生物降解功能。

基于HRP标记抗体的黄曲霉毒素M1的直接竞争-ELISA快速检测方法

为构建快速检测乳制品中黄曲霉毒素M1(AFM1)的直接竞争-酶联免疫吸附(dc-ELISA)体系,将辣根过氧化物酶(HRP)与高纯度抗AFM1单克隆抗体进行偶联后对其与AFM1竞争性反应条件进行优化,并利用AFM1污染标准物质ERMI-BD282(0)、ERMI-BD 283(0.11μg/kg)、ERMI-BD 284(0.44μg/kg)等对检测体系的灵敏度和精确性进行验证。结果当AFM1-BSA包被质量浓度0.25μg/L、抗AFM1 mAb-HRP稀释2000倍时dc-ELISA检测AFM1的IC50为0.75μg/L,检测范围为0.015～4.05μg/L,添加AFM1至0.45μg/L的鲜奶样品中检测回收率平均为80%,dc-ELISA可满足鲜乳中AFM1国家残留限量标准0.5μg/kg的检测要求。该体系可应用于鲜奶制品中AFM 1大于0.5μg/kg污染样品的快速初筛。

黄曲霉毒素M1检测ELISA条件的优化及应用研究

采用黄曲霉毒素M1与牛血清白蛋白偶联物抗原免疫Balb/C小鼠获得特异性抗体。利用此抗体,进行间接竞争ELISA法试验,确定了最佳抗原包被反应浓度为1.0μg·mL-1,最佳抗原包被条件为4℃过夜,一抗和酶标二抗IgG-HRP最佳工作浓度为1∶8000和1∶5000。建立间接竞争定量ELISA方法,该方法的最低检出限是0.08ng·mL-1,间接竞争ELISA50%抑制率浓度为0.62ng·mL-1,校正曲线的线性范围为0.04～5ng·mL-1。在此优化条件下制备的抗体检测黄曲霉毒素M1具有很高的特异性和灵敏性,具有实际应用价值。

牛奶黄曲霉毒素M1的污染状况、风险评估及消减技术研究进展

牛奶黄曲霉毒素M1(aflatoxin M1,AFM1)是由于泌乳奶牛采食被黄曲霉毒素B1(aflatoxin B1,AFB1)污染的日粮,其被机体吸收后经肝脏代谢转化形成的具有致癌性的毒素,是牛奶安全性评价的重要指标。牛奶AFM1污染普遍,传统的牛奶加工方法无法消除AFM1,于是人们近年研究出一些有效降低牛奶AFM1含量的新方法,可为防控牛奶AFM1的污染提供新思路。文章综述了牛奶AFM1的污染状况、风险评估以及牛奶AFM1消减技术的研究进展,以期为牛奶AFM1污染的防控提供参考。

生鲜乳中黄曲霉毒素M1在不同储存条件下的稳定性研究

本文通过检测不同储存条件下生鲜乳中黄曲霉毒素M1(AFM1)的残留水平来分析AFM1的储存稳定性,储存条件包括储存温度、储存时间、复温温度、解冻次数和添加防腐剂等。研究结果表明,在不同的储发存条件下,包括4℃冷藏保存,-20℃冷冻保存,-20℃冷冻保存24h经不同温度复温、反复冻融次数以及添加不同的防腐剂对生鲜乳中AFM1的残留量均无显著影响。当生鲜乳解冻次数不断增加时,检测出AFM1的回收率略呈上升趋势,但结果变化不明显。不同储存条件下的回收率检测值均在80%~110%范围内。

饲料中黄曲霉毒素B1对牛奶黄曲霉毒素M1影响与防控

黄曲霉毒素B1、黄曲霉毒素M1具有极强"三致"作用,污染饲料、乳及乳制品,对奶牛业存在潜在的危害,给人类健康造成严重威胁。饲粮中AFB1转化为乳中AFM1呈现一定的规律,即日粮中AFB1浓度为10μg/kg时,牛奶中会出现AFM10.17μg/kg。降低牛奶AFM1浓度最根本措施是减少饲料中AFB1的污染,防止饲料AFB1污染可以通过控制饲料含水量及防止储存地点污染等措施,饲料中AFB1脱毒解毒方法有物理法、化学法和生物学法等。

酶联免疫法检测牛奶中黄曲霉毒素M1的应用

黄曲霉毒素M1所带来的乳品安全一直是人们关心的话题,测定黄曲霉毒素M1的方法有薄层色谱法、高效液相色谱法、酶联免疫吸附测定法、质谱法、放射免疫测定法等,其中酶联免疫检测法具有特异性强、灵敏度高、分析时间短、成本低的优点,对酶联免疫法检测牛奶中黄曲霉毒素M1的原理、程序、注意事项等进行综述。

免疫亲和-荧光光度法快速测定乳脂及其制品中的黄曲霉毒素M1

本文研究应用荧光光度法测定高乳脂乳和乳制品中的黄曲霉毒素M1,方法检测限为0．1μg8/kg,相对标准偏差低于5%,回收率88%～93%。方法简便快速、灵敏准确,能够满足进出口乳及乳制品中黄曲霉毒素M1的测定。

黄曲霉素M1间接竞争ELISA的建立及其在牛奶中的应用

利用高特异性单克隆抗体,建立黄曲霉素M1的间接竞争ELISA检测方法。棋盘法优化了包被抗原及酶标抗体的最佳稀释倍数。包被抗原和酶标抗体的最佳工作浓度分别为1∶15000和1∶2000。该方法的线性范围为(0.1—8.1)ng·mL-1。除了与黄曲霉素B1的交叉反应为35%外,与黄曲霉素B2、黄曲霉素G1及黄曲霉素G2的交叉反应均小于1%。在牛奶实际样品检测的回收率均大于70%,而且检测过程在10min以内即可完成。该方法灵敏度高,特异性强,而且操作简单,适合多样本的快速筛查,为黄曲霉素M1的高效检测提供了一个良好的选择。

时间分辨荧光免疫层析法检测酥油中黄曲霉毒素M1的研究

为寻求酥油中黄曲霉毒素M1快速准确检测方法,本研究利用时间分辨荧光免疫层析法测定酥油中黄曲霉毒素M1含量,结果表明:该检测方法样品加标回收率为77. 6%89. 6%,RSD <15%,与免疫亲和柱净化-HPLC法相比,相对误差<12%,该法具有简便快速、灵敏度高、重现性好等特点,可用于酥油中黄曲霉毒素M1的快速检测。