Agilent 1100 series chromatograph

The freeze-dried initial WPH, peptide recovery fraction and final WPH lactose content was analyzed by high-performance liquid chromatography (HPLC) [23 ]. Briefly, to perform the analysis, 0.2 g of the sample was solubilized in HPLC-grade water before being treated with Biggs–Szijarto solution. The solution was then centrifuged (5000× g, 5 min, 10 °C). The supernatant was recovered and diluted 10X in HPLC-grade water before being filtered with a 0.45 mm nylon filter. For the HPLC measurements, an Agilent 1100 Series chromatograph (Santa Clara, CA, USA) equipped with an Agilent 1260 Infinity refractive index detector (Santa Clara, CA, USA), a column oven and a cooled 717 Plus autosampler were used. The previously prepared samples were injected onto an ICSep-ICE-ION-300 column (Transgenomic, Omaha, NE, USA), and the mobile phase was a solution of H₂SO₄ (180 μL/L) at a flow rate of 0.4 mL/min. The column was kept under a constant temperature of 40 °C, and the run time was 45 min. To perform the quantification, a standard of lactose anhydrous (Sigma Company, Saint-Louis, MO, USA) was used.

Optical rotations were determined on a PerkinElmer 343 polarimeter (Waltham, MA, USA). IR spectra were recorded using an Equinox 55 spectrophotometer (Bruker, Bremen, Germany). ¹H and ¹³C NMR spectra were recorded on a Bruker Avance III 700 spectrometer (Bruker BioSpin, Bremen, Germany) at 700.13 and 176.04 MHz, respectively, with chemical shifts referenced to the respective residual solvent signal (δ_H 7.21/δ_C 123.5 for C₅D₅N). The HRESIMS spectra were recorded on a Bruker Impact II Q-TOF mass spectrometer (Bruker, Bremen, Germany); the samples were dissolved in MeOH (at 0.001 mg/mL). HPLC separations were carried out on an Agilent 1100 Series chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a differential refractometer; the columns used were as follows: Diasfer-110-C18 (10 µm, 250 × 15 mm, Biochemmack, Moscow, Russia), Discovery C₁₈ (5 µm, 250 × 4 mm, Supelco, North Harrison, PA, USA), and YMC-Pack Pro C18 (5 μm, 250 × 4.6 mm, YMC Co., Ltd., Kyoto, Japan). Low-pressure liquid column chromatography was carried out on Polychrome 1 (powdered Teflon, 0.25−0.50 mm; Biolar, Olaine, Latvia), Florisil (60–100 µm, Sigma-Aldrich Co., St. Louis, MO, USA), and Si gel KSK (50–160 µm, Sorbpolimer, Krasnodar, Russia) columns. Sorbfil Si gel plates (4.5 × 6.0 cm, 5–17 µm, Sorbpolimer, Krasnodar, Russia) were used for thin-layer chromatography (TLC).

Optical rotations, Perkin-Elmer 343 polarimeter (PerkinElmer, Waltham, MA, USA). NMR spectra, Bruker Avance III 700 spectrometer (Bruker BioSpin, Bremen, Germany) at 700.13 MHz (¹H)/176.04 MHz (¹³C), internal standard CD₃OD at δ_H 3.30/δ_C 49.0. HRESIMS spectra, Bruker Impact II Q-TOF mass spectrometer (Bruker, Bremen, Germany); sample concentration in MeOH 0.001 mg/mL. HPLC, Agilent 1100 Series chromatograph (Agilent Technologies, Santa Clara, CA USA) with a differential refractometer; columns Discovery C18 (5 µm, 10.0 × 250 mm, Supelco, Bellefonte, PA, USA) and YMC-Pack Pro C18 (5 µm, 10.0 × 250 mm and 4.6 × 250 mm, YMC Co., Ltd., Kyoto, Japan). LPLC, column sorbents Polychrom 1 (powdered Teflon, 0.25–0.50 mm, Biolar, Olaine, Latvia), Si gel KSK (50–160 µm, Sorbpolimer, Krasnodar, Russia), and Florisil (60–100 µm, Sigma-Aldrich, Co., St. Louis, MO, USA).