运用气相色谱串联质谱法测定健康蜜蜂、中毒蜜蜂和可疑蜂药.结果表明:中毒蜜蜂中含有禁用的克螨特、蝇毒磷及双甲脒代谢物,最高浓度达到7920 μg/kg.可疑蜂药中检出禁用蜂药成分克螨特、蝇毒磷,最高浓度达到9250 mg/kg.分析了蜜蜂中毒可能的原因,提出了科学使用蜂药的建议.

建立了胶体金免疫层析法快速测定蜂胶及蜂胶乙醇提取物中氯霉素残留的检测方法.样品在碱性条件下溶解稀释,20％高氯酸溶液除杂过滤后,加入碱化剂适量,经乙酸乙酯提取,盐析净化后,再用乙酸乙酯提取吹干,加入正己烷和复溶液溶解后,用胶体金快速检测试剂板快速检测.结果表明,胶体金免疫层析法对蜂胶及蜂胶提取物中氯霉素残留量的检测限分别为0.1μg/kg和0.2μg/kg.经高效液相色谱-串联质谱确证,假阳性率和假阴性率均为0％.该方法操作简单、灵敏度高、准确可靠,可用于加工蜂胶及蜂胶乙醇提取物过程中对氯霉素残留量的质量控制.

Summary Royal jelly(RJ)is the exclusive food for the Western honeybee(Apis mellifera)queen throughout its lifespan and the primary food for the honeybee larvae during the first three days of life,which is secreted by hypopharyngeal and mandibular glands of nurse honey bees.The chemical composition of RJ is very complicate, while the protein components have been found to play critical roles in RJ.Around 50% of dry RJ is made up of proteins which could be divided into two parts,water-soluble proteins and water-insoluble proteins.The water-soluble proteins,accounting for 82% to 90% of the total RJ proteins,are named major royal jelly proteins(MRJPs) which belong to the same family composed of nine members.It has been proved that MRJPs have effects such as anti-aging,immunoregulation and anti-fatigue,et al.In addition,MRJPs,especially MRJP1 are authenticated as the critical constituents to reveal the freshness of RJ and will be hopeful as one new functional component in future. In previous studies,various methods were developed for isolation of MRJPs,which included centrifugation, alkali extraction acid precipitation and column chromatography.However,a series of obstacles,such as low purity, solvent contamination and low productivity limited the application of those methods in honeybee industry. Ultrafiltration as a novel bio-separation technology has many advantages,such as simple operation,easy scale-up, and hence has a huge potentiality for large-scale isolation of bio-macromolecules.Our effort was to obtain the separation technology of MRJPs by using ultrafiltration and provide new knowledge for the industrial application of RJ process in future. Since the molecular masses of MRJPs are in the range of 4.9×104 8.7×104,the poly-sulfone hollow fiber membrane component used in the ultrafiltration equipment was comprised of two types with different molecular mass cut-off membranes.The ultrafiltration membrane with a 1 × 105 molecular mass cut-off was employed to remove macromolecular impurities,whereas the 4.9 × 104 cut-off membrane was employed to retain most of MRJPs.In this study,disposals were taken to separate soluble proteins,MRJPs from RJ by ultrafiltration.The control process included pH,ion intensity and ratio of water to material.In order to obtain the optimal conditions to extract MRJPs,orthogonal test was employed during investigation on experimental factors. The results showed that the optimal condition was as follows,ratio of water to material 5∶1 ,ion intensity 0.5 mol/L and pH 7.0.Under the optimal condition,82.63% of MRJPs was extracted from fresh RJ.It was determined that the lyophilized powder of MRJPs contained 91.00% soluble protein,0.01% moisture,2.00% 10-HDA and 15.22% total sugar.Besides,the by-products contained 10-HDA and total sugar were recycled,and the recycling rate of the two components reached 3.60% and 2.11%,respectively. This method with many advantages,such as no solvent pollution and fully utilization of 10-HDA,has provided a new technology for MRJPs separation by first using ultrafiltration in deep process of RJ,and will be hopeful for large scale application in RJ process industry.

Major royal jelly proteins(MRJPs) were hydrolyzed with pepsin and trypsin,and the effects of substrate concentration,pH value,enzyme response time and proteinase concentration on the hydrolysis efficiency were analyzed,and the orthogonal test analysis of combined action of pepsin and trypsin were done.The enzymolysis products of MRJPs(H-MRJPs) were separated to prepare the angiotensin I-converting enzyme(ACE) inhibitory peptides by ultrafiltration technique.The results showed that the optimized technical parameters were as follows:enzymatic hydrolysis of MRJPs with 1% pepsin at pH 2.0 for 2 h,then 1% trypsin at pH 7.5 for 2 h at 37 ℃.The degree of hydrolysis and total nitrogen recovery of MRJPs under the optimal condition were 28.7% and 35.5%,respectively.No obvious protein bands were shown for enzymolysis products of MRJPs(H-MRJPs) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Three kinds of ACE inhibitory peptides,ranging from 1,1-5 and5 ku,were separated by ultrafiltration separation of H-MRJPs.The 50% inhibitory concentration(IC50) values of the three kinds of ACE inhibitory peptides were 0.33,0.61,and 1.09 mg·mL-1,respectively,which exhibited that the peptides with molecular mass 1 ku possessed most high ACE inhibitory activity.The results above provide a scientific basis for developing functional food of antihypertension.

The royal jelly（RJ）is the critical environment factors of caste determination,becoming worker or queen in development for honeybee larvae.Main royal jelly proteins（MRJPs）are the important quality index revealed RJ fresh level.According to Kamakura’s new discovery with honey bee and Drosophila as models,MRJP1 was the critical factors of caste determination in honeybee.The appearance of the characters of queen,such as increasing of body weight,decreasing of developmental time and ovary development appeared were caused by activating an epidermal growth factor receptor（Egfr）signaling pathway in fat body.Therefore,it was essential to further study the nutritional functions and mechanism of MRJP1 in human in future,which will provide knowledge for the protein to be applied in functional foods.