Ultimate 3000 rs

The 12 herbs were mixed and boiled twice with 8 times water (volume/weight) for 30 min each, filtered, and concentrated. 200 μL RBP solution was dissolved in 1 mL methanol and vortexed for 10 min. Then the mixture was centrifuged at 20000 × G for 10 min and filtered through a 0.22-μm microporous membrane. The main ingredients of RBP were measured by ultrahigh-performance liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS) (UltiMate 3000 RS, Thermo Fisher Scientific, USA). The precise details are given in Supplementary File 3.

GDP profiling was performed as previously reported [17 (link)]. Briefly, α-dicarbonyls were derivatized with o-phenylenediamine yielding their respective quinoxaline derivatives. The pH of the double-chamber bag model was adjusted to 5.5 prior to derivatization. A Thermo Fisher UltiMate 3000RS liquid chromatography system consisting of a pump with degasser, autosampler, column compartment, and diode array detector equipped with an ACQUITY UPLC® BEH phenyl column (1.7 μm particle size; 2.1 × 100 mm, Waters, Eschborn, Germany) was used for the chromatographic separation of the quinoxalines. The system was controlled by Chromeleon 6.80 (Thermo Fisher Scientific, Dreieich, Germany). The quinoxaline derivatives were analyzed between 120 and 650 min after adding the derivatizing reagent.

Ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) analysis was performed on a Thermo Fisher UltiMate 3000 RS [Thermo Fisher Scientific (China) Co., Ltd., China] system coupled to a Q-Exactive high-resolution mass spectrometer [Thermo Fisher Scientific (China) Co., Ltd., China]. Two microliters of sample solution was injected into a Thermo Hypersil GOLD column (100×2.1 mm, 1.9 μm). After optimizing the chromatographic conditions, the column temperature was adjusted to 35 °C, and the autosampler was conditioned at 10 °C. A 200 μl sample solution was injected into the system for analysis. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B). A linear gradient elution was applied (0–5 min, 2–20% B; 5–10 min, 20–50% B; 10–15 min, 50–80% B; 15–25 min, 80–95% B; 25–30 min, 95–2% B) at a flow rate of 0.3 ml/min. Mass spectrometric detection was carried out with an electrospray ionization (ESI) interface set in positive or negative ionization mode.

Volatile carboxylic acids in cheese were esterified with ethanol, and analyzed by gas chromatography as described by Fröhlich-Wyder et al. (2013) (link) using a Hewlett Packard HP 6890 gas chromatograph (Agilent Technologies, Basel, Switzerland) equipped with a Hewlett Packard Ultra 2 cross linked phenyl methyl silicone fused silica capillary column (50 m, 0.32 mm, 0.52 mm) and a flame ionization detector (FID). Biogenic amines in cheese were analyzed as described by Ascone et al. (2017) (link) using a UPLC system (UltiMate 3000 RS; Thermo Fisher Scientific, Reinach, Switzerland) equipped with a C18 column (Accucore C18: 2.6 mm, 150 × 4.6 mm; Thermo Fisher Scientific, Reinach, Switzerland). All measurements were carried out in duplicate.

The morphology of RD@MBs was observed using an optical microscope (Leica, Germany). The loading efficiency of R837 and DTX in the MBs were evaluated by Liquid chromatography–mass spectrometry (LC–MS, TSQ Quantum and UltiMate 3000 RS, Thermo Scientific, USA).

Transmission electron microscopy (TEM) images were captured by a Talos F200X G2 (Carl Zeiss SUPER-X, Germany). Scanning electron microscope (SEM) images were recorded on a GeminiSEM 300 (Carl Zeiss SUPRA 55, Germany). Size and zeta potential were analyzed by Malvern zetasizer (ZEN3690, UK). The x-ray diffraction (XRD) pattern was recorded on Ultma IV (Rigaku, Japan) at 45 kV and 200 mA. X-ray photoelectron spectroscopy (XPS) was carried out on K-Alpha (Thermo Fisher Scientific, USA). Fourier transform infrared spectrometry spectra were obtained by a Vertex 70 (Bruker, Germany). Thermogravimetric (TG) analysis was performed on a star simultaneous thermal analyzer (Mettler, Swiss). Surface area tested by surface area and porosity analyzer (Micromeritics ASAP 2460, USA). High-performance liquid chromatography was recorded on an UltiMate 3000 RS (Thermo Fisher Scientific, USA).

Before the UPLC analysis, the ZEA in the growth medium was extracted and cleaned up using an IAC-SEP^® ZEA-specific immunoaffinity column (Clover Technology Group, Beijing, China) according to the manufacturer’s instructions. The resulting sample was transferred to an autosampler vial, and 100 μL was injected into the UPLC column for analysis.
The ZEA content was tested following the Chinese standard method GB 5009.209-2016 [35 ]. Quantitative analysis was performed on a Dionex Ultimate 3000 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) consisting of an Ultimate 3000 RS autosampler, pump, column oven, and fluorescence detector, and controlled by Chromeleon 7.2 software. Separation was performed on a Zorbax Eclipse XDB-C18 RRHD column (100 × 2.1 mm, 1.8 μm; Agilent). The isocratic mobile phase was acetonitrile/water/methanol (46:46:8, v/v) with a flow rate of 1.0 mL/min. The fluorescence detection excitation wavelength was 274 nm, and the emission wavelength was 440 nm.
The ZEA was identified and quantified via comparison with the retention time and the chromatographic peak area of an external standard. The standard curve was obtained by preparing standard solutions at six ZEA concentrations (5, 10, 50, 100, 200, and 500 ng/mL), and each concentration was analyzed in triplicate. The resulting regression equation for ZEA was as follows: y = 2912.3x − 6081.6 (R² = 0.9998).