Sample preparation and analysis by NMR spectroscopy

NMR samples were prepared by weighing out and thawing 2–3 g of frozen powder from the three pools of 50 fruits prepared as described above, and then vortexing and sonicating the resulting juice for 15 min in a cold water bath. The raw juice was centrifuged for 10 min at 15,000 g and 4°C, and then the supernatant was centrifuged for 20 min at 18,000 g and 4°C. We then diluted 0.56 ml of the aqueous extract with buffer to a final volume of 0.7 ml containing 150 mM potassium phosphate, 5% D₂O (Cambridge Isotope Laboratories, Cambridge, UK), 0.02% NaN₃ and 1 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid-d6 (DSS-d6) (Sigma–Aldrich, St Louis, MO, USA). The pH of samples was measured with a mini-electrode and adjusted to pH 6.

One-dimensional ¹H-NOESY spectra were acquired from the aqueous extracts at 25°C using a Bruker Avance III 600 MHz NMR spectrometer (Bruker, Karlsruhe, Germany) equipped with a triple resonance TCI cryogenic probe and operating at a ¹H Larmor frequency of 600.13 MHz. Data were acquired automatically using IconNMR software and an automatic sample changer (both from Bruker). A time delay of 5 min was set between sample injection and pre-acquisition calibrations to allow for complete temperature equilibration. The experiments were recorded with 64 free induction decays (FIDs), 64,000 data points, a spectral width of 20 ppm, 10 s of recycle delay, and a mixing time of 100 ms. The water signal was suppressed using a solvent saturation scheme.

NMR spectra were processed using Topspin v3.2 (Bruker). FIDs were multiplied by an exponential weighting function with line broadening of 0.3 Hz before Fourier transformation, phasing and baseline correction. Processed spectra were referenced to the DSS-d6 singlet signal. Metabolites were annotated using the Profiler module of Chenomx NMR Suite v8.0 (Chenomx, Alberta, Canada) combined with reference compound spectra deposited in the Biological Magnetic Resonance Bank (http://www.bmrb.wisc.edu/). Quantification was achieved by the integration of NMR signals using the DSS-d6 as an internal standard, but for overlapping signals such as fructose we used the Chenomx software instead. The resulting concentrations were corrected for the dilution factor and converted into milligrams per 100 grams of fresh fruit based on the initial recorded weights and volumes. The performance of the NMR laboratory in terms of fingerprinting and quantitative multicomponent analysis was recently assessed in a large-scale inter-laboratory comparison (quality control parameter for the laboratory, QP << 2.0) [29].