A single honey proteome dataset for identifying adulteration by foreign amylases and mining various protein markers...

Honey adulteration is a common practice that deceives consumers and devalues the unique curative and food properties of honey. For marketing, each honey must satisfy an internationally valid Codex standard. One of the quality parameters is diastase/amylase activity, which, if lowered, may be compensated for by the addition of foreign amylases. However, the estimation of enzyme activity does not enable identification of artificially added amylases. 45 honey samples were analyzed using label-free nanoLC-MS/MS proteomics. Four honeys were found to contain the foreign amylases from Aspergillus niger, Bacillus amyloliquefaciens and/or Bacillus licheniformis. This result was confirmed via proof of specificity at multiple levels. Furthermore, we identified a series of plant-related protein groups. Despite plant-related proteins constituting a significant portion of honey proteins, they were minor components compared to the major honey bee-derived proteins. Bioinformatic analysis also provided evidence for aphid and catalase proteins in honey, but the limited specificity of the MS/MS identified peptides must be considered. Overall, we demonstrate a proteomics approach employing LC-MS/MS that is useful for proving adulteration and assessing honey quality. As an resource useful for reference, we provide curated sequence databases. In addition, we provide many markers that are naturally found in honey for future studies. SIGNIFICANCE: Honey is unique natural product used since ancient times as a food and natural medicine. Humans strive to understand honey components because they can characterize different types of honey and be used for authentication and origin assessment. One of the important honey components are proteins. The proteins present in honey can naturally occur in honey, but some of them can be used to mask deficiencies in some honey quality properties. Diastases/amylases are such proteins, and their activity, a measure of honey freshness, can decrease in time or due to processing. To our knowledge, we for the first time specifically identify foreign amylases in honey. However, this study provided new information on other non-honey bee proteins in honey. Thus, this study is also of importance due to its identification of plant and aphid proteins and catalase-related proteins. This study provides a clue explaining the controversial presence of catalase in honey, since catalases can be identified and their origin determined via proteomics.