Straightforward monitoring of honey with foreign diastase by leveraging the differentiation in LC-UV proteome profiles of authentic and fraudulent samples

Abstract

Honey adulteration using sugar syrups is ubiquitous and adulterated honey may also be prepared by direct addition of α-amylases from different sources to match the legislation for honey trade. Colorimetric diastase assays may report false negatives in terms of foreign diastase (FD) detection owing to using substrates being prone to be cleaved by any α-amylases. The main objective of this study was to develop a reliable analytical method to determine FD contained honey. For this purpose, intrinsic honey proteins were first isolated, enriched, and cleaned up via experimentally designed serial ultrafiltration methodology. Next, diastase and invertase as abundant honey enzymes were purified simultaneously from obtained crude protein isolate by means of FPLC and novel purification protocol. Total protein and enzyme activity assays along with SDS-PAGE analysis were conducted for purity check. Subsequently, two purity-confirmed enzymes were used as analytical references in method development. With the hyphenation of UV detection, optimized chromatographic resolution, and practical dilute & shoot sample pretreatment, Foreign Amylase Monitoring (FAM) method has been introduced for routine analysis. Purified authentic enzymes were labeled in LC-UV chromatogram of honey proteome profile to distinguish any unnatural enzyme/protein signal. According to the validation of the FAM method, precise (RSDR; 1.27%), and linear (mean R2 >0.995) results were able to obtain. Commercial honey samples (n = 202) collected over 3 consecutive years were probed using the FAM method and industrial α-amylases were also analyzed for verification. The diastase activities were also measured prior to FAM application to elucidate if any positive correlation. The developed method rapidly and reliably detected the presence of FD qualitatively. A total of 74 samples were found to contain FD. It was observed that the presence of FD was correlated in samples with abnormally high diastase activities and an apparent FD peak was identified readily in all enzyme adulterated samples. The developed FAM method and performed trend analysis allowed us to decipher a dramatic increase in FD existence and this revealed the recently emerging problem of honey authenticity.