Express test for honey quality

Abstract

The following enzymes are found in honey: α- and β-amylase, α- and β-glucosidase, glucose oxidase, catalase, acid phosphatase, glucose dehydrogenase. The glucose oxidase (GOx), which oxidizes glucose in honey, produces gluconic acid, one of the main acids found in honey, and generates hydrogen peroxide, which is an important antimicrobial component of honey. The more hydrogen peroxide, resulting from higher GOx activity, is found, the higher the value of honey is. This work was aimed to evaluate the activity of GOx in honey samples collected in different regions of Lithuania Hydrogen peroxide is produced in honey in the process of glucose oxidation by GOx, as one of the reaction’s products. It also acts as a substrate for peroxidase accepting protons. Due to the high peroxidase selectivity we developed an express test for honey quality evaluation. The test is based on peroxidase immobilized on a silica gel beads and coloured substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). The carrier is selected from a number of inorganic and organic carriers (activated carbon, hydrated alumina, polystyrene). Peroxidase immobilized on this carrier had a higher enzymatic activity and temperature stability. The developed method was applied for the measurement of GOx activity in 10-fold dilute samples of honey until 0.01 activity units/mL. The method is well correlated with the reference method, which is based on the measurement of GOx activity by an oxygen consumption rate (R=0.978). The express method is a visual test of honey quality in a set of green colour palette that corresponds to certain GOx concentration in the examined sample. The method allows us to produce dry kits and perform honey quality tests without sophisticated analytical techniques at room temperature. The test is about 10 min, the resulting colour is compared with the colour palette, which shows enzyme concentration in the sample. The dry kit is stable for up to 4 months at 4 ° C. This is especially convenient for beekeepers in order to prove the exceptional quality of the bee products they market. The study found that the GOx activity of Lithuanian honey varied within 0.03 – 1.49 U/g range. The highest GOx activity was identified in buckwheat and caraway honeys, while the lowest GOx activity was in oilseed rape and alder buckthorn honeys. The test could also help detect overheated or adulterated honey.