Chemical and peptide profiling for New Zealand mānuka (Leptospermum scoparium) honey authentication

Abstract

New Zealand mānuka (Leptospermum scoparium) honey is internationally recognised for its unique non-peroxide antibacterial activity. Parallel with the industry efforts in establishing a robust and reliable guideline for defining New Zealand mānuka (L. scoparium) honey, two alternative approaches for honey authentication based on chemical and peptide profiling were examined. Leptosperin was identified as the fluorophore responsible for the unique fluorescence characteristic of mānuka honey at ex270-em365 nm (MM1). Chemical analyses based based on four key attributes, namely honey concentration, nectar origin, chemical stability, and ease of detection, identified leptosperin and lepteridine as the most definitive chemical markers for mānuka honey authentication. 2-Methoxybenzoic acid and 2'-methoxyacetophenone were also found to be unique to mānuka honey. However, the concentrations of these compounds appear to be unstable over the honey's shelf-life. 3-Phenyllactic acid and 4-hydroxyphenyllactic acid are relatively stable in honey but not mānuka-specific, being shared with the co-harvested kānuka (Kunzea ericoides). A solid-phase fluorimetric EZQ® assay of total protein content in monofloral New Zealand honeys demonstrated similar protein content of <0.2% w/w except for ling (Calluna vulgaris) honey, which contained approximately three-fold greater protein content. A novel peptide profiling approach based on shotgun proteomics was also investigated using nanoLC-QqTOF-MS/MS. The protein assemblage of mānuka honey was described for the first time comprising bee- and plant-derived proteins. The mānuka proteins were identified based on the predicted mānuka proteome derived from Leptospermum 'Crimson Glory' (supplied by Plant and Food Research Ltd.). A total of twelve nectar-derived mānuka peptide markers were identified, eight of which exhibited 100% matching sequence identity to L. 'Crimson Glory' (PM1-PM8), and the remaining four represented either sequence variant or deamidated form of these L. 'Crimson Glory' peptides. A targeted proteomic method based on parallel reaction monitoring (PRM) was developed for relative quantification of the mānuka peptide markers in honey. These peptide markers unambiguously distinguished mānuka from the other New Zealand honey types. Extension of the study to the Australian honey crops demonstrated peptide profiling as a promising approach to distinguish New Zealand and Australian Leptospermum honeys, including the L. scoparium var. eximium honey harvested in Tasmania.