Diphenylamine

For all cultivations performed, samples were taken in regular intervals. The OD₆₀₀ was measured with a spectrophotometer (Biochrom WPA CO8000, Biochrom Ltd., Cambridge, United Kingdom). The cell dry weight was calculated by dividing the OD₆₀₀ by the factor 3.762 which was determined previously (Geissler et al., 2019b (link)). Prior to further analyses, samples were centrifuged for 10 min at 4°C and 4816 g (Heraeus X3R, Thermo Fisher Scientific GmbH, Braunschweig, Germany) and stored at −20°C until further processing.
Glucose was measured with a HPTLC system (CAMAG AG, Muttenz, Switzerland) as described in Geissler et al. (2019b) (link). Briefly, the mobile phase used was acetonitrile/H₂O (85:15, v/v) and plates were developed over a migration distance of 70 mm. After development, plates were derivatized with diphenylamine (DPA) reagent. DPA was prepared by diluting 2.4 g diphenylamine and 2.4 g aniline in 200 mL methanol and then adding 20 mL 85% phosphoric acid. After derivatization, plates were scanned at 620 nm and the glucose concentration was calculated in dependence of the standard curve.
Surfactin analysis was performed as described in Geissler et al. (2017) (link) using a HPTLC method. Briefly, samples were extracted three times with an equal volume of chloroform:methanol 2:1 (v/v). The pooled solvent layers were evaporated and the crude surfactin was resuspended in methanol to match the initial sample volume. Plates were developed using the mobile phase chloroform:methanol:water 65:25:4 (v/v/v) over a migration distance of 60 mm. After development, the plates were scanned at 195 nm and evaluation was performed by peak area in correspondence to a standard curve.
Spectrophotometric assays (Merck KGaA, Darmstadt, Germany) were used to measure nitrate (Cat. No. 1.09713.0001), nitrite (Cat. No. 1.14776.0001) and ammonium (Cat. No. 1.14752.0001) concentrations.
Acetate concentration was determined with an enzymatic kit (Cat. No. 10148261035, r-biopharm AG, Pfungstadt, Germany).
For ß-galactosidase assay, a volume of 100 μL of cell suspension from strain MG1 or MG5 was mixed with 900 μL Z-Buffer (0.06 mol/L Na₂HPO₄, 0.04 mol/L NaH₂PO₄, 0.01 mol/L KCl, 1 mmol/L MgSO₄ ⋅ 7 H₂O, 0.04 mol/L mercaptoethanol). After addition of 10 μL toluol, the mixture was incubated for 30 min at 37°C and 750 rpm. 200 μL of 20 mmol/L ortho-nitrophenylgalactopyranoside was added and the reaction was stopped when the solution turned yellow by adding 500 μL of 1 mol/L Na₂CO₃. Samples were centrifuged for 2 min at 19283 g and 250 μL were transferred to a microtiter plate. Absorbance was measured at both 420 nm and 550 nm and the Miller Units (MU) were calculated according to the following equation:

All standards and samples were applied as 8-mm bands 8 mm from the lower edge of the HPTLC plate using a semiautomated HPTLC application device (Linomat 5, CAMAG, Muttenz, Switzerland) set at a speed of 50 nLs⁻¹. The glucose, fructose, sucrose and maltose standard curves were obtained by applying 1 µL, 2 µL, 3 µL, 4 µL and 5 µL of the respective standard solutions (calibration curves available in the Supplementary Material section). For the analysis of sugars in the honey, syrup and adulterated honey samples, 3 µL of each sample solution were applied.
The chromatographic separation was performed at ambient temperature on silica gel 60 F₂₅₄ HPTLC plates (glass plates 20 × 10 cm) in a saturated (33% relative humidity) automated development chamber (ADC2, CAMAG). The development chamber was saturated for 60 min, and the HPTLC plates were presaturated with mobile phase (1-butanol: 2-propanol: aqueous boric acid (5 mg/mL) 30:50:10 v/v/v) for 5 min. The plates were automatically developed to a distance of 85 mm, and after drying for 5 min they were analyzed under white light using an HPTLC imaging device (TLC Visualizer 2, CAMAG). The chromatographic images were digitally processed using specialized HPTLC software (visionCATS, CAMAG) [27 (link),28 (link),32 (link)].
After documentation of the initial chromatographic results, the HPTLC plates were derivatised with 2 mL of the aniline-diphenylamine-phosphoric acid reagent (CAMAG Derivatiser). After heating for 10 min at 115 °C (CAMAG TLC Plate Heater III) and cooling to room temperature, the plates were re-analysed under transmission white (T white) light using the HPTLC imaging device.

Concerning the techniques employed in the analysis of the samples, we used gas chromatography (GC) coupled with mass spectrometry (GS/MS) with electronic ionisation and acquisition in selected ion monitoring. The GC/MS system was a GC 7890 from Agilent Technologies interfaced with an Agilent 5975 MS, with an HP5 column (17 m × 0.2 mm × 0.33 μm). The parameters for GC were the following: oven programme – 85 °C (1 min), 15 °C/min to 270 °C, 50 °C/min to 310 °C (3.5 min); injection – 1 µL, pulsed splitless; injector temperature – 270 °C. We followed the protocol that is normally used for narcotics and stimulants [10 (link)]. The urine samples were alkalinised with NaOH and NaCl was added for a salting out effect. The samples were extracted with tert-butyl methyl ether. The organic extracts were then taken to dryness under a reduced nitrogen flow at room temperature and reconstituted with an extraction solvent before GC/MS analysis. Diphenylamine was added as an internal standard (ISTD).
The estimated nicotine concentration was > 50 ng/mL according to the WADA monitoring program, which is based on allowing for the lower limit of quantification and a conservative concentration limit for active consumption [4 (link)]. The advantage of this conservative limit is that active consumption is clearly distinct from passive exposure (e.g. tobacco smoke) and trace amounts through diet (e.g. potatoes, cauliflower) in ‘tobacco-free’ individuals, as well as abstinent nicotine users [3 (link), 4 (link)]. However, a disadvantage is a potential under-estimation of true positivity.