Multinuclear z gradient inverse probehead

The NMR experiment was done as described elsewhere by Sobolev et al. (2014) (link). Briefly, samples for NMR were prepared by dissolve in 5–10 mg of an extract in a deuterated solvent (methanol-d₄ or the mixture of acetone-d₆/D₂O). The NMR spectra of extracts were recorded at 27 °C on a Bruker AVANCE 600 NMR spectrometer operating at the proton frequency of 600.13 MHz and equipped with a Bruker multinuclear z-gradient inverse probe head. ¹H spectra were acquired by adding 128 transients with a recycle delay of 3 s. The experiments were carried out by using a 90°pulse of 10 µs, 32K data points.

Aliquots of kefir samples were analyzed by NMR spectroscopy in order to assess their chemical composition. In particular, the assignment of the resonances was performed by analyzing ¹H characteristics and cross-correlated signals in 2D ¹H-¹H TOCSY spectra and by comparison with literature data [22 (link),25 (link),26 (link)]. Quantification of the identified compounds was performed by comparison of the signal integral with the reference one, and quantities were expressed in mg of compound normalized for the aliquot weight expressed in g. Each dry aliquot was dissolved in 0.6 mL of D₂O:CD₃OD (2:1 ratio) containing 3-(trimethylsilyl)-propionic-2,2,3,3-d₄ acid sodium salt 2 mM as chemical shift and concentration reference. All spectra were recorded at 298 K on a Bruker AVANCE III spectrometer operating at the proton frequency of 400.13 MHz and equipped with a Bruker multinuclear z-gradient inverse probehead. ¹H spectra were acquired employing the presat pulse sequence for solvent suppression with 128 transients, a spectral width of 6000 Hz and 64 K data points for an acquisition time of 5.45 s. The recycle delay was set to 6.55 s in order to achieve complete resonance relaxation between successive scansions. ¹H–¹H TOCSY experiments were acquired with spectral width of 6000 Hz in both dimensions, a data matrix of 8K × 256 points, mixing time of 110 ms and relaxation delay of 2 s.

All spectra were recorded at 298 K on a Bruker AVANCE III spectrometer (Bruker BioSpin, Karlsruhe, Germany), equipped with a Bruker multinuclear z-gradient inverse probe head operating at the proton frequency of 400.13 MHz. More details can be found in Supporting Information (Extended Materials and Methods).

The NMR spectra of aqueous and organic extracts were recorded at 27 °C on a Bruker AVANCE 600 NMR spectrometer operating at the proton frequency of 600.13 MHz and equipped with a Bruker multinuclear z-gradient inverse probe head capable of producing gradients in the z-direction with a strength of 55 G/cm. ¹H spectra were referenced to methyl group signals of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP, δ = 0.00 ppm) in D₂O and to the residual CHD₂ signal of methanol (set to 3.31 ppm) in CDCl₃/CD₃OD mixture [53 (link)]. ¹H spectra of aqueous extracts were acquired by coadding 512 transients with a recycle delay of 3 s. The residual HDO signal was suppressed using a standard Bruker presaturation sequence zgpr. The experiment was carried out by using a 45° pulse of 7.25 μs and 32,000 data points. ¹H spectra of CDCl₃/CD₃OD extracts were obtained using the following parameters: 256 transients, 32,000 data points, a recycle delay of 3 s, and a 90° pulse of 10 μs. The ¹H spectra were Fourier transformed using an exponential multiplication function with a line broadening factor of 0.3 Hz, and manual phase correction and baseline correction were applied.