Mining foreign proteins in honey by shotgun label-free proteomics: Implications for quality and authenticity

Honey may contain various foreign proteins, most notably from protein and carbohydrate supplements or from artificial additions to mask enzyme activity. Here, we analyzed 29 honey samples and one royal jelly sample, which served as a control alongside "our" own production-verified honeys. We also reanalyzed a previous dataset of 45 honey samples. We found that the honey samples in both datasets contained different "foreign" enzymes used for syrup production and/or as bee fondant. The accuracy of their identification is supported by the large number of unique peptides obtained for each protein. These include Aspergillus glucoamylase (γ-amylase), Saccharomyces invertase, and α-amylase. The results also suggest that Aspergillus catalase, used as a highly efficient antioxidant, is a foreign enzyme. Furthermore, we identified the presence of soy seed storage proteins such as glycinin and conglycinin. The presence of these proteins alongside foreign enzymes indicates either poor beekeeping practices regarding supplemental feeding or the intentional use of supplemental feeding. We also report the presence of plant- and human-derived proteins in honey. In summary, our study broadens the usefulness of shotgun proteomics in identifying the various proteins in honey that can be used to determine its composition, quality, and authenticity. SIGNIFICANCE: Despite the use of sophisticated methods such as NMR, HRMS, and IRMS, honey is one of the most adulterated foods, and fraud is widespread. One of the useful methods for solving the problem of honey fraud is proteomics. This study demonstrates the ability of proteomics to identify proteins that should not be present in honey. These "foreign proteins" can indicate honey fraud and/or bad beekeeping practices, resulting in low honey quality. The results suggest that a group of these proteins are enzymes used in the industrial production of syrup. These enzymes include α-amylase, glucoamylase (γ-amylase), and invertase (β-fructofuranosidase). Additionally, proteomics can be used to detect proteins such as glycinin/conglycinin and lipoxygenase from soybean seeds in honey. Their identification suggests that the bees were fed protein supplements, the residues of which are found in honey. All these proteins should not be present in high-quality honey. This study also identified other proteins that were not previously reported in honey. In particular, these is an array of human-derived proteins whose identification is consistent with the fact that beekeeping, honey harvesting, and processing are not sterile activities. Identifying human proteins is scientifically interesting because it helps us understand honey properties and can be used in database searches. Overall, analysis using discovery/untargeted proteomics can identify proteins of interest in honey, including those associated with fraud, quality issues, or botanical origin. One specific group of interest is plant-derived proteins.