Pavlović Ratko, Stojanović Sanja, Pavlović Marija, Drulović Nenad, Vujčić Miroslava, Dojnov Biljana, Vujčić Zoran
Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia.
Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njegoševa 12, 11000 Belgrade, Serbia.
Insects. 2025 Jun 12;16(6):622. doi: 10.3390/insects16060622.
The enzymes in honey can originate not only from bees and the plants from which the bees collect pollen and nectar but also from feed provided by beekeepers. Enzymes that hydrolyze sucrose-present in honey (α-glucosidase) or honey adulterated with invert syrup (β-fructofuranosidase)-can be distinguished using zymography, where enzymatic bands are detected with nitroblue tetrazolium (NBT) after sugar removal via ultrafiltration. This method enables the identification of honey produced in hives that have been improperly fed with invert syrup, leading to the mixture of natural honey and syrup, and offers a practical tool to detect indirect adulteration. The NBT assay, in combination with ultrafiltration, was used to determine the isoelectric point of honey bee α-glucosidases. The pI value of 6.63 for isoforms found in the head, midgut, and natural honey extracts during winter can be attributed to α-glucosidase III. Two additional isoforms with isoelectric points of 5.20 and 5.77 were observed in the midgut extract and may correspond to α-glucosidase I and II. The difference between α-glucosidase and β-fructofuranosidase was confirmed using a substrate specificity test, followed by thin-layer chromatography, where it was confirmed that α-glucosidase from natural honey, bee head, and bee midgut does not hydrolyze raffinose, in contrast to yeast β-fructofuranosidase.
蜂蜜中的酶不仅可以源自蜜蜂以及蜜蜂采集花粉和花蜜的植物，还可以源自养蜂人提供的饲料。可以使用酶谱法区分水解蜂蜜中存在的蔗糖的酶（α-葡萄糖苷酶）或掺有转化糖浆的蜂蜜中的酶（β-呋喃果糖苷酶），在通过超滤去除糖分后，用硝基蓝四氮唑（NBT）检测酶带。这种方法能够鉴定用转化糖浆不当饲喂的蜂巢中生产的蜂蜜，这种蜂蜜会导致天然蜂蜜与糖浆混合，并且提供了一种检测间接掺假的实用工具。NBT测定法与超滤相结合，用于确定蜜蜂α-葡萄糖苷酶的等电点。冬季在头部、中肠和天然蜂蜜提取物中发现的同工型的pI值为6.63，可归因于α-葡萄糖苷酶III。在中肠提取物中观察到另外两种等电点分别为5.20和5.77的同工型，可能对应于α-葡萄糖苷酶I和II。使用底物特异性测试，随后进行薄层色谱法，证实了α-葡萄糖苷酶和β-呋喃果糖苷酶之间的差异，其中证实天然蜂蜜、蜜蜂头部和蜜蜂中肠中的α-葡萄糖苷酶不水解棉子糖，这与酵母β-呋喃果糖苷酶不同。
