Kromasil c18 column

The reaction mixture containing an oligopeptide (0.5 mg/mL) and trypsin (5 μg/mL) in phosphate buffered saline (PBS, pH 7.4) was incubated for different time intervals, and aliquots of digestion products were collected for reversed-phase high-performance liquid chromatography (HPLC). HPLC analysis was carried out on a Dionex UltiMate 3000 system (Thermo Scientific) using a Kromasil C18 column, 5 μm, 4.6 mm × 150 mm (AkzoNobel). Mobile phase contained (A) acetonitrile with 0.1% trifluoroacetic acid, (B) milli-Q water with 0.1% trifluoroacetic acid. Gradient scheme was as follows, 0–10 min: from 100% A to 100% B, 10–20 min: 100% A to 100% B, 20–25 min: 100% B. Flow rate was 0.5 mL/min; injection volume was 10 μL. The detection was performed at wavelengths of 220 and 260 nm. Chromatographic data were collected and treated with the aid of Chromeleon 6.80 software (Thermo Scientific). Peak height of the uncleaved oligopeptides was measured to calculate remaining concentration of the oligopeptides after tryptic cleavage. Concentration values were presented as mean ± SD (n = 3).

The CH MPs association efficiency was determined upon the separation of MPs from the aqueous preparation medium containing the non-associated protein by centrifugation (15,000× g, 45 min, 15 °C). The amount of free peptide was determined in the supernatant by a HPLC-UV (Waters Alliance^® instrument (Milford, MA, USA)) method. In this method, a Kromasil^® C18 column (AkzoNobel, Bohus, Sweden) was used and the UV detector wavelength was set to 280 nm. The mobile phases consisted of acetonitrile and 0.1% TFA, and water and 0.1% TFA. The ratio was initially set at the ratio of 80:20 (acetonitrile: 0.1% TFA, v/v), which linearly changed to a 40:60 (v/v) gradient over 10 min. The flow rate was 0.8 mL/min and the injected volume of the sample was 20 μL. The UV detector wavelength was set at 280 nm. The total area under the peak was used to quantify the KGYGGVSLPEW peptide sequence. Each sample was assayed in triplicate (n = 3). The CH MPs peptide association efficiency (AE) and loading degree (LD) were calculated as follows (Equations (1) and (2)): $AE (\%)=\frac{total peptide amount-free peptide amount}{total peptide amount}\times 100,$
$LD (\%)=\frac{total peptide amount-free peptide amount}{peptide loaded CH MPs dry weight}\times 100,$

The effect of bacterial cell-free culture supernatants on the DON production of F. graminearum was observed by wheat kernels DON assay according to Reddy et al. [19 (link)] with some modifications. About 10 g of healthy and dry wheat kernels (purchased locally, and the water content was 0.9%) were mixed with 2 mL of bacterial cell-free culture supernatants and 1 mL of conidia suspension in a 100 mL conical flask. In the control flask, bacterial culture supernatants were replaced by deionized water. For each bacterial strain and the control, there were three replicated flasks. After incubation at 25 °C for 18 days, the wheat kernels were ground by a grinder. Twenty-five mL of 84% methanol in water was added, and the mixture was shaken for 2 h, and then centrifuged at 7,155 g for 10 min. The solid residue was re-extracted, and the supernatants were combined. Before the analysis with HPLC, using Varian ProStar (Varian, Palo Alto, CA, USA) connected with UV-VIS detector (190–700 nm), the supernatant was filtered through 0.22 μm polyvinylidene fluoride (PVDF) syringe filter. The chromatographic column was a Kromasil C18 column (250 mm × 4.6 mm, 5 μm, Akzo Nobel, Bohus, Sweden). The mobile phase was 80% methanol in water and the flow rate was 0.8 mL·min⁻¹, with the wavelength of detection at 218 nm. Peak area in the chromatogram was used to represent the relative DON content.