Multi-Platform Chemical Fingerprinting for Authentication and Characterisation of New Zealand Monofloral Honeys

Abstract

This research developed and evaluated an evidence-chain framework for authenticating New Zealand monofloral honeys (thyme, mānuka, kānuka and clover) while linking compositional fingerprints to functional bioactivity. As melissopalynology and single-marker testing can be inconclusive, especially for the closely related Myrtaceae honeys (mānuka/kānuka), this research tested whether combining orthogonal analytical platforms with interpretable modelling could produce defensible, class-consistent drivers suitable for routine screening. An intentionally unbalanced design (thyme n=13; mānuka/kānuka/clover n=3 each), reflecting thyme’s commercial relevance and strong preliminary bioactivity, was used to stress-test robustness under realistic sampling constraints. Twenty-two raw and commercial samples were characterised using (i) HS-SPME-GC-MS volatile profiling, (ii) a dual-window ¹H NMR strategy (direct honey profiling and C18-SPE enrichment to mitigate sugar-matrix suppression), (iii) targeted compositional panels (sugars, minerals, phenolics and selected bioactive constituents), and (iv) bioactivity assays (FRAP, CUPRAC, DPPH and anti-tyrosinase). Across chapters, PLS-DA, multinomial elastic net, Random Forest with Shapley Additive Explanations (RF-SHAP) and multiblock integration (DIABLO and sPLS regression) were implemented with sample-level stratified cross-validation and repeated resampling. Candidate marker sets were further constrained by requiring dominant target-class abundance (highest median), a minimum mean positive SHAP threshold and class-consistency support (positive SHAP across all target-class samples) to reduce outlier-driven findings.

HS-SPME-GC-MS delivered rapid discrimination with near-ceiling performance (5-fold micro-average ROC-AUC 0.995) and chemically interpretable drivers consistent with floral origin: thyme was characterised by oxygenated terpenoids and related aroma chemistry, clover by distinctive aldehydic/benzenoid features, and the Myrtaceae honeys by overlapping but separable plant-derived signatures. Direct-honey ¹H NMR, although dominated by the carbohydrate manifold, still achieved strong out-of-fold separability (micro-AUC ≈0.98) and highlighted regions consistent with established mānuka chemistry (including low-ppm and mid-field features aligned with MGO/DHA-associated resonances), while thyme was supported by aromatic windows around 7.42–7.38 ppm. C18-SPE NMR increased dynamic range (micro-AUC ≈0.99) and revealed enriched aromatic/phenolic regions that strengthened interpretability and cross-platform convergence.

Integrating targeted sugars, minerals, and phenolics translated complex chemistry into audit-ready decision rules: PLS-DA, elastic net and RF-SHAP achieved strong discrimination (ROC-AUC ≈0.95-0.99) and resolved botanically coherent drivers. Lumichrome emerged as a robust kānuka-associated discriminant, mānuka aligned with a Leptospermum-type profile including DL-3-phenyllactic acid, and thyme showed a coordinated antioxidant-leaning signature spanning bioactive constituents and composition (vitamin C/ascorbic acid, syringic acid and sucrose).

Finally, multiblock integration linking composition to function demonstrated a coherent chemico-biological gradient: Welch ANOVA confirmed honey-type effects across all bioactivity endpoints (p ≤ 0.004), with thyme highest and clover lowest, while mānuka/kānuka were intermediate and partially overlapping. DIABLO and sPLS regression indicated that the high-activity phenotype reflected a convergent multi-domain signature involving vitamin C, sucrose, phosphorus and phenolic acids, whereas lumichrome contributed mainly to genus-level discrimination within the Myrtaceae profile. Overall, the thesis shows that authenticity is best supported by convergent multi-platform signatures rather than single biomarkers, and provides a reproducible, interpretable workflow for New Zealand monofloral honey authentication and bioactivity interpretation.