Honey adulteration, where a range of sugar syrups is used to increase bulk volume, is a common problem that has significant negative impacts on the honey industry, both economically and from a consumer confidence perspective. This paper investigates High-Performance Thin Layer Chromatography (HPTLC) for the authentication and detection of sugar adulterants in honey. The sugar composition of various Australian honeys (Manuka, Jarrah, Marri, Karri, Peppermint and White Gum) was first determined to illustrate the variance depending on the floral origin. Two of the honeys (Manuka and Jarrah) were then artificially adulterated with six different sugar syrups (rice, corn, golden, treacle, glucose and maple syrup). The findings demonstrate that HPTLC sugar profiles, in combination with organic extract profiles, can easily detect the sugar adulterants. As major sugars found in honey, the quantification of fructose and glucose, and their concentration ratio can be used to authenticate the honeys. Quantifications of sucrose and maltose can be used to identify the type of syrup adulterant, in particular when used in combination with HPTLC fingerprinting of the organic honey extracts.

The antibacterial activity of honeys derived from the endemic flora of the southwest corner of Western Australia, including the trees Jarrah (Eucalyptus marginata) and Marri (Corymbia calophylla), remains largely unexplored. Investigation of these honeys showed minimum inhibitory concentrations (MICs) of 6.7–28.0% (w/v) against Gram positive and negative bacteria. Honey solutions showed enhanced antibacterial activity after hydrogen peroxide was allowed to accumulate prior to testing, with a mean MIC after accumulation of 14.3% compared to 17.4% before accumulation. Antibacterial activity was reduced after treatment with catalase enzyme, with a mean MIC of 29.4% with catalase compared to 15.2% without catalase. Tests investigating the role of the Gram negative outer membrane in honey susceptibility revealed increases in activity after destabilisation of the outer membrane. Honeys reduced both the formation of biofilm and the production of bacterial pigments, which are both regulated by quorum sensing. However, these reductions were closely correlated with global growth inhibition. Honey applied to existing biofilms resulted in decreased metabolic activity and minor decreases in viability. These results enhance our understanding of the mechanisms of antibacterial action of Jarrah and Marri honeys, and provide further support for the use of honey in the treatment of infected wounds.

This study reports on the total phenolic content and antioxidant activity as well as the phenolic compounds that are present in Calothamnus spp. (Red Bell), Agonis flexuosa (Coastal Peppermint), Corymbia calophylla (Marri) and Eucalyptus marginata (Jarrah) honeys from Western Australia. The honey’s total phenolic content (TPC) was determined using a modified Folin–Ciocalteu assay, while their total antioxidant activity was determined using FRAP and DPPH assays. Phenolic constituents were identified using a High Performance Thin-Layer Chromatography (HTPLC)-derived phenolic database, and the identified phenolic compounds were quantified using HPTLC. Finally, constituents that contribute to the honeys’ antioxidant activity were identified using a DPPH-HPTLC bioautography assay. Based on the results, Calothamnus spp. honey (n = 8) was found to contain the highest (59.4 ± 7.91 mg GAE/100 g) TPC, followed by Eucalyptus marginata honey (50.58 ± 3.76 mg GAE/100 g), Agonis flexuosa honey (36.08 ± 4.2 mg GAE/100 g) and Corymbia calophylla honey (29.15 ± 5.46 mg GAE/100 g). In the FRAP assay, Calothamnus spp. honey also had the highest activity (9.24 ± 1.68 mmol Fe²⁺/kg), followed by Eucalyptus marginata honey (mmol Fe²⁺/kg), whereas Agonis flexuosa (5.45 ± 1.64 mmol Fe²⁺/kg) and Corymbia calophylla honeys (4.48 ± 0.82 mmol Fe²⁺/kg) had comparable FRAP activity. In the DPPH assay, when the mean values were compared, it was found that Calothamnus spp. honey again had the highest activity (3.88 ± 0.96 mmol TE/kg) while the mean DPPH antioxidant activity of Eucalyptus marginata, Agonis flexuosa, and Corymbia calophylla honeys were comparable. Kojic acid and epigallocatechin gallate were found in all honeys, whilst other constituents (e.g., m-coumaric acid, lumichrome, gallic acid, taxifolin, luteolin, epicatechin, hesperitin, eudesmic acid, syringic acid, protocatechuic acid, t-cinnamic acid, o-anisic acid) were only identified in some of the honeys. DPPH-HPTLC bioautography demonstrated that most of the identified compounds possess antioxidant activity, except for t-cinnamic acid, eudesmic acid, o-anisic acid, and lumichrome.

The use of High-Performance Thin-Layer Chromatography (HPTLC) coupled with the use of DPPH* (2,2-diphenyl-1-picrylhydrazyl) as a derivatisation reagent is a novel approach to the analysis of antioxidant activity of honeys. The method facilitates the visualisation of individual constituents that contribute to the overall antioxidant activity of the honey, even if they are not yet chemically identified, and allows for the quantification of their antioxidant activity as gallic acid equivalents. The method supports a more in-depth study of the antioxidant activity of honey as it allows for a comparative analysis of the antioxidant fingerprints of honeys of different floral origin and is able to capture differences in their individual bioactive constituents. Further, it supports the tracking of changes in antioxidant activity of individual honey constituents over time upon exposure to different temperature conditions, which demonstrates the potential value of the method for in-process quality control.

This study reports on the analysis of eleven Jarrah (Eucalyptus marginata) honeys, of which nearly half (n = 5) were re-classified as Blackbutt (E. patens) honey on the grounds of the predominant flower pollen identified by melissopalynology. Based on a comprehensive analysis of the honeys' physico- and phytochemical characteristics and antioxidant activity data, taking into account pH, electrical conductivity, refractive index and Brix values as well as moisture content, individual fructose and glucose content and derived fructose to glucose ratio alongside total phenolic content and antioxidant activity determined by the DPPH assay, no statistically significant difference was found amongst the eleven honeys classified by pollen analysis into two honey groups, ‘Jarrah’ or ‘Blackbutt’. This study therefore draws into question the value of melissopalynology as an analysis tool to authenticate Jarrah honey.

High-Performance Thin-Layer Chromatography (HPTLC) was used in a chemometric investigation of the derived sugar and organic extract profiles of two different honeys (Manuka and Jarrah) with adulterants. Each honey was adulterated with one of six different sugar syrups (rice, corn, golden, treacle, glucose and maple syrups) in five different concentrations (10%, 20%, 30%, 40%, and 50% w/w). The chemometric analysis was based on the combined sugar and organic extract profiles’ datasets. To obtain the respective sugar profiles, the amount of fructose, glucose, maltose, and sucrose present in the honey was quantified and for the organic extract profile, the honey’s dichloromethane extract was investigated at 254 and 366 nm, as well as at T (Transmittance) white light and at 366 nm after derivatisation. The presence of sugar syrups, even at a concentration of only 10%, significantly influenced the honeys’ sugar and organic extract profiles and multivariate data analysis of these profiles, in particular cluster analysis (CA), principal component analysis (PCA), principal component regression (PCR), partial least-squares regression (PLSR) and Machine Learning using an artificial neural network (ANN), were able to detect post-harvest syrup adulterations and to discriminate between neat and adulterated honey samples. Cluster analysis and principal component analysis, for instance, could easily differentiate between neat and adulterated honeys through the use of CA or PCA plots. In particular the presence of excess amounts of maltose and sucrose allowed for the detection of sugar adulterants and adulterated honeys by HPTLC-multivariate data analysis. Partial least-squares regression and artificial neural networking were employed, with augmented datasets, to develop optimal calibration for the adulterated honeys and to predict those accurately, which suggests a good predictive capacity of the developed model.