2016年国内外蜂产品质量安全研究进展（续）

周金慧 张文文 王欣然 张金振 金玥 杨树鹏 赵文 陈兰珍 问亚琴 王鹏 黄京平 刘婷婷 李熠

（中国农业科学院蜜蜂研究所农业部蜂产品质量监督检验测试中心，北京100093）

2016年国内外蜂产品质量安全研究进展（续）

周金慧 张文文 王欣然 张金振 金玥 杨树鹏 赵文 陈兰珍 问亚琴 王鹏 黄京平 刘婷婷 李熠

（中国农业科学院蜜蜂研究所农业部蜂产品质量监督检验测试中心，北京100093）

（续《中国蜂业》2017年第3期）

重金属分析的样品前处理方法和仪器分析方法也得到了一定的发展。通过合成一种螯合树脂（MPAEMA-co-DVB-co-AMPS）作为吸附剂来提取蜂蜜样品中Cd(II)、Co(II)、Cr(III)、Cu(II)、Fe(III)、Mn(II)、Pb(II)、和Zn (II)离子，然后利用原子荧光光谱法定量分析，检测限范围为0.9～2.2 ng/ml[27]。微波消解-四级杆-电感耦合等离子体质谱也被应用于蜂蜜和蜂花粉中Sc、Y、La、Ce、Pr、Nd、Sm、Eu、Gd、Dy、Ho、Er、Tm、Lu、Yb、As、Bi、Cd、Pb、Se和In含量分析，通过调整四级杆和质谱的参数能够极大地减少蜂蜜和蜂花粉基质影响，提高准确度和灵敏度[28]。

蜂蜜主要成分为糖类物质，包括果糖和葡萄糖在内的所有的糖类物质占80%以上，在高糖的环境下微生物很难存活，但是研究显示芽孢杆菌因为其耐受的芽孢体的存在而广泛存在于蜂蜜中。基于MALDITOF-MS质谱技术对蜂蜜中芽孢杆菌的鉴定与分型显示44株芽孢杆菌中鉴定出蜡样芽孢杆菌 (B.cereus)31株，短小芽孢杆菌 (B.pumilus)5株，枯草芽孢杆菌(B. subtilis)3株，地衣芽孢杆菌(B.licheniformis)2株，炭疽芽孢杆菌 (B.anthracis)1株，土壤短芽孢杆菌(Brevibacillus agri.)2株。通过多次重复试验，表明从同一份样品或同一品牌蜂蜜样品中能稳定分离到芽孢杆菌，获得的蛋白指纹图谱具有极好的稳定性[29]。

2.3 溯源分析

蜂产品尤其是蜂蜜的溯源分析是近年来研究的热点，其研究主要是基于蜂产品理化性质、矿物质元素以及黄酮类、酚酸类、氨基酸和芳香类等内源性活性组分的靶标和非靶标数据统计分析构建模型，使之可视化的分类。

基于矿物元素含量可进行蜂蜜蜜源和产地溯源研究，采用全谱直读电感耦合等离子体原子发射光谱法测定蜜样矿物元素含量，引进哑变量回归和校正模型，从而建立了蜂蜜产地、蜜源双目标溯源分析方法[30]。基于蜂蜜中的氨基酸种类和含量的不同，应用甲醛和乙酰丙酮与蜂蜜中氨基酸发生荧光衍生反应对源自不同花源的蜂蜜进行种类辨别研究。对衍生后的蜂蜜进行三维荧光检测，每个蜂蜜样品经检测后得到一个三维荧光光谱矩阵数据，五种蜂蜜共150个样品最后得到一个三维立方数据。将检测获得的三维荧光数据结合多维主成分分析、自加权交替三线性分解法及多维偏最小二乘辨别分析等多维模式识别方法，进行数据处理并获取五种蜂蜜的识别信息。研究结果显示，基于氨基酸荧光衍生的多维模式识别方法可以用于蜂蜜种类的识别研究[31]。曹炜等[32]基于高效液相色谱-电化学检测技术建立一种新的蜂蜜花源鉴别方法，以采自中国不同地区的3种单花种蜂蜜为研究对象，构建了3种单花种蜂蜜的液相色谱电化学检测器指纹图谱，提取图谱共有峰面积信息并应用主成分分析和系统聚类分析进行蜂蜜花源分类，并对完全未参与建模的蜂蜜样品进行验证。此外世界各国的研究人员还分别利用蜂蜜中的理化指标、特征组分寻找、内源性组分结合多种数据分析手段对来自不同国家的不同蜜源植物的蜂蜜进行了鉴别[33-46]。

先进的样品前处理技术和仪器分析技术也用于蜂产品的溯源分析。以铅笔铅为机体的铋膜电极结合化学计量学方法，通过优化方波伏安法的参数，使用Behnken设计与期望函数相结合，不同的算法如不对称最小使用正方形（AsLS）和相关优化变形（COW）来预处理原始像数据，偏最小二乘法（PLS）和人工神经元网络（ANN）用来预测样品中四种金属的含量。该方法最终用来鉴别阿根廷不同产地的蜂胶产品[47]。高效液相薄层色谱-荧光检测器和实时直接分析质谱指纹图谱也用于法国蜂胶的鉴别，获得的指纹图谱数据利用模式识别技术对大量数据进行分析，同时酚酸类化合物咖啡酸、对香豆酸、柯茵、短叶松素、3-O-乙酰基短叶松素、高良姜素、莰菲醇和松属素作为鉴别法国蜂胶的特征标志物[48]。

大气压分析探针质谱是近两年出现的又一种环境大气压离子化质谱分析技术，该技术能够快速、简便分析固体、液体、组织或材料样品中挥发性和半挥发性化合物而无需样品制备和分离。探针插放于商品化的质谱离子源的源体内，热的氮气流使样品快速脱附至空气中，经电晕放电离子化产生质子化(正离子模式)或去质子化(负离子模式)离子，随后进行质谱或多级质谱定性、定量分析。不干扰同一源体上的电喷雾(ESI)源或大气压化学电离源（APCI）源的运行，互为补充，且切换迅速方便。该离子化方式尤其适用于高端液质平台，充分展示其多级质谱的选择性碰撞碎裂能力，进行快速鉴定和高灵敏度定量，以及精确质量分析能力，实现复杂混合物中化合物的快速鉴定和定量分析。该技术在蜂花粉鉴别种也得到了应用，蜂花粉磨碎后借助于微量的水附着于玻璃毛细管上，然后涂于探针，通过提高氮气的温度使花粉中的物质至离子源附近，在低碰撞能量条件下的质谱中分析，从而通过分析黄酮类物质鉴别花粉的蜜源植物和产地[49]。

2.4 掺假鉴别

目前，世界各国的蜂产品掺假主要集中于蜂蜜和蜂胶两个产品。我国蜂蜜国家标准规定，不得在蜂蜜中添加或混入任何淀粉类、糖类或代糖类物质。近年来，国内和国际市场对蜂蜜的需求量不断增加，在巨大经济利益的驱使下不法分子在蜂蜜中加入其他低品质的蜂蜜以次充好，或者掺入糖浆等甜味物质以假乱真，影响了蜂蜜产品的市场秩序和我国蜂蜜产品的出口贸易。而蜂胶被定义为工蜂采集植物树脂等分泌物与其上颚腺、蜡腺等分泌物混合形成的胶粘性物质。由于其独特而广泛的活性作用，因此市场需求量很大，进而也就出现了杨树胶混入蜂胶中的造假问题。蜂胶来源于植物树脂，尤其是杨树蜂胶主要来源于杨属植物，因此为鉴别蜂胶和杨树胶带来了难度。

蜜蜂在采集杨属植物树脂以及蜂巢内传递这些树脂加工成蜂胶的过程中加入了其腺体分泌的β-葡萄糖苷酶等，将树脂中所含有的水杨苷水解，但在杨树胶加工过程中水杨苷能稳定存在，因而水杨苷是区分蜂胶与杨树胶的有效指标。基于液相色谱-线性离子阱-静电场轨道阱高分辨质谱快速测定蜂胶中杨树胶指标性成分水杨苷含量的方法，能够根据精确的母离子和子离子质荷比进行定性和确证，以判断蜂胶中是否掺杂杨树胶[50]。

根据AOAC 998.12的描述，蜂蜜中C-4糖含量＞7%被认定为掺假蜂蜜。但是，按照该方法测定C-4糖含量＜0%的蜂蜜样品真实性的研究较少。使用元素分析仪和液相色谱法电感耦合同位素质谱分析蜂蜜及其提取的蛋白质和糖（蔗糖，葡萄糖和果糖）中δ13C值，δ2H和δ18O值，以及蔗糖和还原糖含量，进而研究C-4糖含量＜0%的蜂蜜样品的真实性。研究结果显示，与0＜C-4糖含量（%）＜7相比，蜂蜜在-7＜C-4糖含量（%）＜0时被鉴定为不含C-4糖更加可靠。对于δ18O值，和其他的蜂蜜相比，C-4糖含量（%）＜-7组值较低为16.30‰，这可以作为一个鉴别掺假蜂蜜有用参数。该项研究表明，使用同位素组分和系统偏差能够可靠的检测C-4糖含量＜0%的蜂蜜样品是否掺假[51]。除了添加糖浆之外，一些不法商贩也在蜂蜜中添加防腐剂、甜味剂、色素和香精等外源性添加剂，陈丽娟等[52]将蜂蜜样品采用甲醇-水溶液提取苯甲酸、山梨酸、安赛蜜、糖精钠，以乙酸铵溶液和甲醇为流动相梯度洗脱，通过高分辨质谱负离子扫描模式进行定性，外标法定量，有效考察苯甲酸、山梨酸、安赛蜜和糖精钠在蜂蜜中添加情况。

3.总结

2016年已经正式出版的文献主要侧重于蜂产品中待测物的快速、简单的样品前处理技术的改进和建立高分辨质谱技术的应用，进而提高待测物免受基质干扰的能力，提高待测物的灵敏度和准确度。尤其是电化学技术、新材料技术、免疫胶体金技术和利用分析化学技术在原有质谱基础上的改进相结合的应用。

本文仅列举了部分具有代表性的相关文献，以点带面阐述2016年本领域内的研究概况，希望对以后研究内容的范围和深度都有所帮助。

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[22]郑明,江明,胡卫南,等.原子吸收光谱法和原子荧光光谱法测定蜂产品中的重金属残留量 [J].中国现代应用药学,2016, (10):1297-1300.

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[26]张剑,王祯旭,李秀梅.微波消解-电感耦合等离子质谱法测定蜂胶制品中5种重金属元素的含量[J].中国药业,2016,25 (5):52-54.

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