黄曲霉对不同含水量新疆鲜食核桃活性氧代谢的影响

以新疆“新2”薄皮核桃为材料,无菌水接种为对照组,探究人工接种从自然霉变核桃上分离纯化出的黄曲霉菌后不同含水量的鲜食核桃活性氧代谢相关酶的变化规律。结果表明,不同含水量的鲜食核桃遭受到黄曲霉侵染后,在侵染前期,各含水量的鲜食核桃体内活性氧迅速累积,酶促清除系统被激活,不同的抗氧化酶为抵御病原微生物的侵染活性开始有明显的升高并达到峰值,但随着侵染时间的延长,酶促清除系统清除活性氧的能力减弱,活性氧的大量积累导致果实细胞膜结构被破坏,加快了其老化、腐烂。黄曲霉侵染含水量25%的鲜食核桃时,酶促清除系统的抗氧化酶活性明显高于其他含水量的鲜食核桃。     

含水量对玉米粉贮藏期黄曲霉生长及黄曲霉毒素B1积累的影响

目的 研究含水量对玉米粉贮藏过程中黄曲霉生长及黄曲霉毒素B1积累的影响。方法 将初始水分含量10.42%、13.26%、16.35%、19.88%的玉米粉在28 ℃, 相对湿度75%的模拟环境中储藏30 d; 测定贮藏期间玉米粉含水量、黄曲霉生长量及黄曲霉毒素B1的积累量。结果 贮藏期间玉米粉中含水量呈现先上升后下降的变化趋势, 黄曲霉生长量及黄曲霉毒素B1的积累量随贮藏时间的延迟先快速升高, 后趋于平稳。初始含水量较高的玉米粉贮藏期间含水量、黄曲霉生长量及黄曲霉毒素B1含量始终处于较高的水平。相关性分析表明, 玉米粉含水量、黄曲霉生长量和毒素积累量之间均呈极显著的正相关(P<0.01)。结论 含水量是影响玉米粉贮藏过程中霉菌生长和毒素积累的关键因素, 降低玉米粉初始含水量可以有效减轻贮藏期间真菌危害。     

乳与乳制品中黄曲霉毒素M1研究进展

目的：建立基于不同序列长度适配体的纳米金（Gold nanoparticles,AuNPs）比色传感法快速定量检测牛奶中的黄曲霉毒素M₁（Aflatoxin M₁,AFM₁）,并评价目前文献报道中常用的21（A21）和72个碱基（A72）长度的AFM₁适配体在实际样品中的检测性能。方法：采用柠檬酸钠还原法制备AuNPs溶液,加入AFM₁适配体及AFM₁标准品后,适配体与AFM₁特异性结合形成特殊三维结构,随着NaCl溶液加入,AuNPs溶液的稳定性被破坏而发生聚集,导致溶液颜色变化,通过测定AuNPs溶液的吸光值和吸收光谱定量检测AFM₁。结果：通过优化适配体浓度、NaCl浓度、反应温度及pH等实验条件,基于适配体A21和A72的AuNPs比色传感法检测AFM₁的检测限分别为25.26和5.77 µg/L,线性范围均为10~800 µg/L,且均具有良好的选择性及特异性,在牛奶中的加标回收率分别为95.7%~103.6%、94.3%~98.2%。结论：A72检测牛奶中AFM₁的效果优于A21,可能与适配体长度、构型及亲和性相关。基于适配体A72建立的比色传感法简单、快速、灵敏、特异性强且可视化,为AFM₁适配体在乳制品中的应用提供数据参考。

     

基于还原氧化石墨烯的电化学适配体传感器对黄曲霉毒素M1的检测

本实验基于还原氧化石墨烯（RGO）构建了一种用于黄曲霉毒素M₁（AFM₁）检测的电化学适配体传感器。采用红枣汁还原氧化石墨烯（GO）制备RGO,RGO通过滴涂法修饰在玻碳电极（GCE）表面,利用电沉积法将纳米金修饰在RGO/GCE上,AFM₁的适配体（Apt）通过Au-S键固定在AuNPs/RGO/GCE电极表面用于靶标AFM₁的捕获。当AFM₁存在时,AFM₁与适配体特异性结合形成AFM₁-Apt复合物,该复合物阻碍了电子的传递,导致电化学信号减弱。对RGO的制备条件进行优化,利用差示脉冲伏安法（DPV）监测电极表面的电化学信号,并对不同类型的毒素（黄曲霉毒素B₁、黄曲霉毒素B₂、赭曲霉毒素A和伏马毒素B₁）、不同浓度的AFM₁（1×10?7~5×10?4 ng/mL）以及羊乳样品进行检测以确定电化学适配体传感器的特异性、灵敏性和实用性。结果表明,GO:红枣汁=2:1（V:V）,pH=11时所制备的RGO的导电能力最强。传感器的电信号与AFM₁浓度的对数呈线性关系,检测范围为1×10?7~5×10?4 ng/mL,检测限为3.3×10?5 pg/mL,同时所建立的方法仅对AFM₁的检测有响应,而对干扰毒素无响应,说明电化学适配体传感器的特异性良好。使用建立的AFM₁电化学适配体传感器对羊奶中的AFM₁含量进行测定,发现所构建的传感器具有很高的灵敏性和良好的选择性,有望应用于食品工业中真菌毒素的快速、准确检测当中。     

乳及乳制品中黄曲霉毒素M1污染及检测技术研究进展

乳及乳制品中的黄曲霉毒素M_1是黄曲霉毒素B1的羟基化衍生物,对肝脏有致畸和致癌作用。随着人们生活水平的提高,乳及乳制品中黄曲霉毒素M_1污染也引起越来越多的关注。本文从不同品种、季节及地域分析乳及乳制品中黄曲霉毒素M_1污染水平,并综述了液相色谱法和免疫法的检测技术研究进展。     

生物技术对黄曲霉毒素B1的脱除及机制研究进展

黄曲霉毒素是危害最大的真菌毒素之一,而黄曲霉毒素B₁(AFB₁)是黄曲霉毒素中毒性最大的一种,具有高毒性、致癌、致畸和致突变的作用,对动物和人类健康造成危害。为了有效脱除AFB₁,综述了近年来研究的具有解毒作用的微生物,介绍了采用生物技术脱除AFB₁的方法,重点探讨了生物脱除AFB₁机制。用于脱除AFB₁的菌株有芽孢杆菌、假单胞菌、乳酸菌、非产毒曲霉等,可采用单菌种发酵、多菌种协同发酵和菌-酶协同作用脱除AFB₁。生物脱除AFB₁机制主要为降解脱除和吸附脱除。可进一步筛选具有高优良能力的菌株以及更深层次地研究微生物脱毒的机制,探索微生物制剂对AFB₁的降解作用。     

牛奶及奶粉中黄曲霉毒素M1的快速测定

采用免疫亲和柱－荧光光度法快速测定了牛乳及乳制品中黄曲霉毒素M1。试样经过离心、脱脂、过滤后滤液经过键合有黄曲霉毒素M1特殊抗体的免疫亲和柱净化，此抗体对黄曲霉毒素M1具有专一的识别能力，黄曲霉毒素M1键合在分离柱中的抗体上，用甲醇与水之比为10：90的混合液将免疫亲和柱上杂质除去；以甲醇与水之比为80：20的混合液通过分离柱洗脱；加入溴溶液衍生，以提高测定灵敏度，衍生化后的洗脱液于荧光光度计中测定黄曲霉毒素M1，检测低限为0．1μg/kg；在0．1－1．0μg/kg范围内，回收率为89．6％－96．1％，变异系数为0．52％－5．8％，分析一个样品的时间小于30min，分析过程中不使用黄曲霉毒素M1标准物质，结果表明，本方法具有准确、简单、快速、安全等优点，可以满足少量和批量样品的检测需要。     

微生物代谢黄曲霉毒素B1的研究进展

黄曲霉毒素是由黄曲霉、寄生曲霉等真菌产生的次级代谢产物,主要存在于谷类、花生、玉米等农作物中。在我国尤其是南方省份,受高温高湿环境的影响,黄曲霉毒素在农作物上的污染极易发生,造成巨大的经济损失。利用微生物代谢黄曲霉毒素,具有专一、高效、反应条件温和等诸多优点,是目前公认的理想脱毒方法。本文首先简要介绍了黄曲霉毒素B₁的形成过程、结构特征以及毒性基团,接着重点介绍了已报道的能代谢黄曲霉毒素B₁的微生物种类、关键酶以及代谢途径,最后列举了应用基础研究的例子。     

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。     

牛奶中黄曲霉毒素的来源与控制

本文概述了牛奶中黄曲霉毒素的存在种类,来源及其性质.对近年来国内外对牛奶中黄曲霉毒素毒素的检测方法、控制和消除方法进行了研究.     

High genetic variability in non-aflatoxigenic A. flavus strains by using Quadruplex PCR-based assay

Aflatoxigenic Aspergillus flavus isolates always show, by using a multiplex PCR-system, four DNA fragments specific for aflR, nor-1, ver-1, and omt-A genes. Non-aflatoxigenic A. flavus strains give variable DNA banding pattern lacking one, two, three or four of these genes. Recently, it has been found and reported that some aflatoxin non-producing A. flavus strains show a complete set of genes. Because less is known about the incidence of structural genes aflR, nor-1, ver-1 and omt-A in aflatoxin non-producing strains of A. flavus, we decided to study the frequencies of the aflatoxin structural genes in non-aflatoxigenic A. flavus strains isolated from food and feed commodities. The results can be summarized as following: 36.5% of the examined non-aflatoxigenic A. flavus strains showed DNA fragments that correspond to the complete set of genes (quadruplet pattern) as found in aflatoxigenic A. flavus. Forty three strains (32%) showed three DNA banding patterns grouped in four profiles where nor-1, ver-1 and omt-A was the most frequent profile. Twenty five (18.7%) of non-aflatoxigenic A. flavus strains yielded two DNA banding pattern whereas sixteen (12%) of the strains showed one DNA banding pattern. In one strain, isolated from poultry feed, no DNA bands were found. The nor-1 gene was the most representative between the four aflatoxin structural assayed genes. Lower incidence was found for aflR gene. Our data show a high level of genetic variability among non-aflatoxigenic A. flavus isolates that require greater attention in order to design molecular experiment to distinguish true aflatoxigenic from non-aflatoxigenic A. flavus strains.     

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.     

Distribution of aflatoxin M1 during Grana Padano cheese production from naturally contaminated milk

The distribution of aflatoxin M₁ (AFM₁) has been investigated in samples of whey, curd and a typical hard and long maturing cheese like Grana Padano (ripened for twelve months), produced with naturally contaminated milk in a range of 30–98 ng AFM₁/kg. AFM₁ determinations were carried out on 25 samples of each product by reverse-phase HPLC and fluorescence detection with post-column derivatisation, after a preliminary C18-SPE clean-up. Experimental results show that, in comparison to milk, AFM₁ concentration levels increased both in curd (3-fold) and in long maturing cheese (4.5-fold), while AFM₁ occurrence in whey decreased by 40%.     

远红外辐照对大米黄曲霉孢子及其产毒能力的影响

以接种黄曲霉孢子的大米为原料,采用远红外辐照杀菌技术,研究了红外辐照对黄曲霉孢子的杀灭效果、生长曲线和产黄曲霉毒素B1(AFB1)能力的影响,同时考察了其对大米的色泽、游离氨基酸、可溶性蛋白等品质的影响。结果表明,远红外对黄曲霉孢子的杀灭效果随着辐照温度的升高和时间的延长而显著增强。当大米含水率为30%时,辐照温度115℃处理5 min,黄曲霉对数降低值(lgS)为2.96±0.28,AFB1总量下降63.09%,单位菌体产毒量下降38.44%;当大米含水率为20%时,lgS为2.04±0.17,AFB1总量下降55.04%,单位菌体产毒量下降22.54%。采用先115℃高温处理5min后70℃保温5 min,大米黄曲霉lgS3.87,L、b、△E和游离氨基酸含量均无显著性差异,可溶性蛋白含量随着处理时间的延长逐渐降低。     

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products

A sensitive and specific monoclonal antibody (Mab) against aflatoxin M₁ (AFM₁), named as 2C9, was selected by semi-solid HAT medium. It exhibited high affinity for AFM₁ of 1.74 × 10⁹ L/mol and no cross-reactivity to aflatoxin B₁, B₂, G₁ and G₂. Based on the antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM₁ in milk and infant milk products. Assays were performed in the AFM₁-BSA coated (0.0625 μg/mL) ELISA format in which the antibody was diluted 1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assay performance were optimised. Finally, the limits of detection were 3 ng/L for milk and 6 ng/L for milk-based cereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged from 91% to 110%. Thirty samples were analysed, and concordant results were obtained when the data were compared with a reference high-performance liquid chromatography method.     

Degradation of aflatoxin B1 by fungal laccase enzymes

The enzymatic degradation of aflatoxin B₁ (AFB₁) by white rot fungi through laccase production was investigated in different liquid media. A significant (P < 0.0001) correlation was observed between laccase activity and AFB₁ degradation exhibited by representatives of Peniophora and Pleurotus ostreatus cultivated in minimal salts (MSM) (r = 0.93) and mineral salts — malt extract (MSB–MEB) (r = 0.77) liquid media. Peniophora sp. SCC0152 cultured in MSB–MEB liquid medium supplemented with veratryl alcohol and sugarcane bagasse showed high laccase activity (496 U/L), as well as 40.45% AFB₁ degradation as monitored using high performance liquid chromatography. P. ostreatus St2-3 cultivated in MSM liquid medium supplemented with veratryl alcohol resulted in laccase activity of 416.39 U/L and 35.90% degradation of AFB₁. Aflatoxin B₁ was significantly (P < 0.0001) degraded when treated with pure laccase enzyme from Trametes versicolor (1 U/ml, 87.34%) and recombinant laccase produced by Aspergillus niger D15-Lcc2#3 (118 U/L, 55%). Aflatoxin B₁ degradation by laccase enzyme from T. versicolor and recombinant laccase enzyme produced by A. niger D15-Lcc2#3 coincided with significant (P < 0.001) loss of mutagenicity of AFB₁, as evaluated in the Salmonella typhimurium mutagenicity assay. The degradation of AFB₁ by white rot fungi could be an important bio-control measure to reduce the level of this mycotoxin in food commodities.     

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.     

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.     

An effective self-control strategy for the reduction of aflatoxin M1 content in milk and to decrease the exposure of consumers

The study reports the results of testing the sensitivity of an early warning sampling plan for detecting milk batches with high aflatoxin AFM₁ concentration. The effectiveness of the method was investigated by the analysis of 9017 milk samples collected in Italian milk processing plants that applied control plans with different action limits (AL). For those milk processing plants where 30 ng kg^?1 AL has been applied, the AFM₁ contamination was significantly lower at or above the 95th percentile of the milk samples when compared with plants that used 40 ng kg^?1 AL. The results show that the control plan can be used effectively for early warning of occurrence of high AFM₁ contamination of milk and to carry out pro-active measures to limit the level of contamination. Estimation of dietary exposure was also carried out, based on the aflatoxin M₁ content of the milk samples and on Italian food consumption data. Estimated Daily Intakes (EDI) and Hazard Indices (HI) were calculated for different age groups of the population. HIs show that no adverse effects are expected for the adult population, but in the case of children under age three, the approximate HI values were considerably higher. This underlines the importance of the careful monitoring and control of aflatoxin M₁ in milk and dairy products.