Authentication of Jarrah (Eucalyptus marginata) honey through its nectar signature and assessment of its typical physicochemical characteristics

Jarrah nectar

Jarrah nectar was collected from Jarrah (Eucalyptus marginata) trees within their known range (Fig. 1B), specifically in Jarrahdale (32.33814°S, 116.06267°E; Date: 29 October 2023), which is located on the Darling escarpment within the Jarrah Forest biogeographical region, and from Margaret River (33.96226°S, 115.02576°E; Date: 14 October 2023) within the Warren region (Department of Agriculture, Water and the Environment, Commonwealth of Australia, 2012). The trees for nectar collection were identified by their flowers, buds and capsules together with their distinct bark (Dell, Havel & Malajczuk, 2012; Hingston, O’Connell & Grove, 1989; Slee et al., 2020). Flowers were obtained in the early morning, stored in a sealed container, and placed in a fridge for transport to the laboratory. Each flower was washed three times with 10 µl of distilled water and the solution collected in an opaque vial, which was stored at 4 °C until analysis.

Jarrah honey

From the southwest of Western Australia 500 samples were collected and a subsample of 31 samples labelled as Jarrah honey (Table 1) were provided by beekeepers between November 2015 and December 2020 from apiary sites within the Jarrah Forest. The samples were stored in glass jars in the dark at room temperature until analysis.

Jarrah honey blend

Thirteen samples were selected as “typical” Jarrah honey samples. A sub-sample of 5 g was batched and mixed to create a blend (‘HPTLC analysis of organic honey and nectar extracts’).

Preparation of honey and nectar samples

For the preparation of nectar and honey organic extracts of approximately 1 g of honey, or a nectar solution, were mixed with two mL of deionised water. The aqueous solution was then extracted three times with five mL of dichloromethane. The combined organic extracts were dried with anhydrous MgSO₄, filtered and the solvent evaporated at ambient temperature. The extract was stored at 4 °C and reconstituted in 100 µL dichloromethane before HPTLC analysis.

For the sugar analysis, 100 mg of honey was dissolved in 80 mL of 50% aqueous methanol and then made up to 100 mL with 50% aqueous methanol.

Standard, mobile phase and reagent preparation

For the HPTLC analysis of the organic honey and nectar extracts, a methanolic solution of 0.5 mg/mL of 4,5,7-trihydroxyflavanone was prepared as a reference standard and a mixture of toluene: ethyl acetate: and formic acid (6:5:1, v/v/v) as mobile phase. The vanillin derivatisation reagent was prepared by dissolving 1 g of vanillin in 100 mL of ethanol, followed by the dropwise addition of two mL of sulphuric acid.

To identify and quantify the honey’s main sugars, standard glucose, fructose, maltose, and sucrose solutions were prepared by dissolving the respective sugar in 50% aqueous methanol. The concentration of standards was fructose (250 ng/µL), glucose (250 ng/µL), maltose (50 ng/µL) and sucrose (100 ng/µL).

A 3:5:1 v/v/v mixture of 1-butanol: 2-propanol: boric acid (5 mg/mL in water) was prepared as a mobile phase. For the derivatisation reagent, 2 g of diphenylamine and two mL of aniline were dissolved in 80 mL of methanol. After the addition of 10 mL of phosphoric acid (85%), the solution was made up to 100 mL using methanol.

For the determination of 5-hydroxymethylfurfural (HMF) content, Carrez solution I was prepared by dissolving 15 g potassium ferrocyanide (K₄Fe(CN)₆. 3H₂O) in 100 ml deionized water. Carrez solution II was prepared by dissolving 30 g zinc acetate (Zn(CH₃CO₂)₂ ⋅2H₂O) in 100 mL deionized water.

Jarrah nectar and jarrah honey organic extracts

The reference standard (4 µL) and the respective nectar (5 uL and 20 uL) or honey (5 uL) organic extract solutions were applied as eight mm bands at eight mm from the lower edge of the HPTLC plate (glass plates 20 × 10 cm, silica gel 60 F254) at a rate of 150 nLs⁻¹ using a semi-automated HPTLC application device (Linomat 5; CAMAG). The chromatographic separation was performed in a saturated and activated (33% relative humidity) automated development chamber (ADC2; CAMAG). The plates were pre-conditioned with the mobile phase for 5 min and automatically developed to a distance of 70 mm at a fixed ambient temperature (Islam, 2022).

The obtained chromatographic results were recorded using an HPTLC imaging device (TLC Visualizer 2; CAMAG) at 254 nm and 366 nm, respectively. After the initial documentation of the chromatographic results, each plate was derivatised with three mL of vanillin-sulfuric acid reagent and heated for 3 min at 115 °C using a CAMAG TLC Plate Heater III. The plate was cooled to room temperature and analysed with the HPTLC imaging device under white light and at 366 nm. The scanning of individual major bands in the nectar and honey extracts, before and after derivatisation, was carried out using a TLC Scanner 4. The chromatographic images were digitally processed and analysed using specialised HPTLC software (visionCATS; CAMAG), which was also used to control the individual instrumentation modules (Islam et al., 2021a; Locher et al., 2018).

Jarrah honey sugar profile

Sugar standard solutions were applied as eight mm bands at eight mm from the lower edge of the HPTLC plate (glass plates 20 × 10 cm, silica gel 60 F254) at a rate of 50 nLs ⁻¹ using a semi-automated HPTLC application device (Linomat 5; CAMAG). To prepare the glucose, fructose, sucrose and maltose standard curves, 1 µL, 2 µL, 3 µL, and 4 µL of the respective standard solutions were applied. To quantify the fructose and glucose content, and maltose and sucrose content of the Jarrah honey blend, 2 µL and 6 µL respectively, of the honey solution was applied.

The chromatographic separation was performed in a saturated (33% relative humidity) automated development chamber (ADC2; CAMAG). The development chamber was saturated for 60 min, and the plates were pre-conditioned with the mobile phase for 5 min, automatically developed to a distance of 85 mm at a fixed ambient temperature and dried for 5 min. The obtained chromatographic results were recorded using an HPTLC imaging device (TLC Visualizer 2; CAMAG) under white light (Islam, 2022).

After the initial documentation of the chromatographic results, each plate was derivatised with two mL of aniline-diphenylamine-phosphoric acid reagent using a TLC derivatiser (CAMAG Derivatiser). The derivatised plates were heated for 10 min at 115 °C using a CAMAG TLC Plate Heater III. The plates were then cooled to room temperature and analysed with the HPTLC imaging device under white light (Islam et al., 2020; Islam et al., 2021b). The chromatographic images were digitally processed and analysed using specialised HPTLC software (visionCATS; CAMAG), which was also used to control the individual instrumentation modules.

Physico-chemical characteristics of jarrah honey

The key physicochemical characteristics such as pH, electric conductivity, Brix value and moisture content, water-insoluble content, free acidity, diastase and 5-hydroxymethylfurfural (HMF) content were determined by standard analytical methodologies following Codex Alimentarius guidelines (Alimentarius, 2017).

In brief, the pH of honey was measured by dissolving 10 g of honey in 75 ml of carbon-dioxide-free water (Meda et al., 2005) and the resulting pH of the solution was determined with a calibrated pH meter (HI98131; Hanna Instruments, Woonsocket, RI, USA).

The electrical conductivity of a 20% (w/v) honey solution was measured at 22 °C using an Electrical Conductometer (HI98131, Hanna Instruments, Rhode Island, USA) and expressed as milliSiemens per centimetre (mS/cm) (Adaškevičiūte et al., 2019).

Brix value and moisture content were determined simultaneously by spreading the honey over the entire surface of the reading window of a digital refractometer (HI96800, Hanna Instruments, Woonsocket, RI, USA). The moisture content of the honey sample was derived from the respective ‘Refractive index (20 °C) vs Moisture content (percent)’ chart (Bogdanov, Martin & Lullmann, 2002).

Water insoluble content was determined by dissolving 10 g of honey into deionised water. The solution was filtered through a previously dried and weighed filter paper (8 µm, No. 540, Whatman Ltd, England, UK). The filter paper was washed thoroughly with hot water (80 °C) until free from sugar before being dried for 60 min at 130 °C, cooled and re-weighed. The difference between prefiltered and postfiltered weight was divided by the mass of honey and expressed as percent water-insoluble solid.

Free acidity was determined by dissolving 10 g of honey in 75 ml of distilled carbon dioxide-free water. The sample solution was titrated against 0.1 N sodium hydroxide solution using 4–5 drops of phenolphthalein indicator (Bogdanov, Martin & Lullmann, 2002). The result was expressed as milli-equivalents acid/kg honey.

Diastase content was determined by following the Phadebas^® Honey Diastase Test. In brief, exactly 1 g of honey was dissolved in 100 ml acetate buffer solution. A five mL sample of this solution was incubated at 40 °C and, after 5 min, one Phadebas tablet was added and vortex mixed. The solution was then incubated at 40 °C for 30 min before adding one mL of 0.5 M sodium hydroxide. The mixture was vortexed and centrifuged at 1,500 G for 5 min before its absorbance was measured at 620 nm (Cary 60 UV-Vis; Agilent, Santa Clara, CA, United States) (Phadebas, 2024; Tosi et al., 2008). A blank without honey was treated in the same manner. The result was expressed as a Diastase Number (DN) from the chart listed in the Phadebas^® Honey Diastase Test user manual.

The 5-Hydroxymethylfurfural (HMF) content of the honey was determined following the White method (White, 1979). In brief, exactly 5 g of honey was dissolved in 25 mL of deionized water. The solution was transferred into 50 mL volumetric flasks and 0.5 mL of Carrez Solution I was added. After mixing, 0.5 mL of Carrez Solution II was added, followed by a thorough mixing. The volumetric flask was made up to 50 mL with deionized water and filtered (Grade 4: 20–25 µm; Whatman Ltd, Little Chalfont, UK). Two samples of five mL of filtrate each were collected in two different test tubes. five mL of deionized water was added to one of the test tubes (sample), whereas five mL of 0.20% sodium bisulfite (NaHSO₃) solution was added to the second test tube (reference). Both test tubes were vortex mixed and the absorbance of the samples measured at 284 nm and 336 nm (Cary 60 UV-Vis; Agilent) with the sample compared against the reference as blank (PerkinElmer, 2023).

The HMF content of honey was calculated using following equation: ${\displaystyle HMF\left(mg/100gHoney\right)=\frac{\left(A_{284}-A_{336}\right)\times 74\cdot 87}{w}}$ Where, w = weight of sample (g)

The HMF content was expressed as mg/kg honey.