Glycosidase Isoforms in Honey and the Honey Bee (Apis mellifera L.): Differentiating Bee- and Yeast-Derived Enzymes and Implications for Honey Authentication.

Abstract

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.