High performance liquid chromatograph

The collected bacterial solution was passed through a 0.22-µm membrane filter and diluted with 10% trichloroacetic acid at a ratio of 1:1, and then 0.5 mL was used for amino acid analysis on a high-performance liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) according to a previously reported protocol [36 (link)].

Mycobacterium bovis BCG was cultured in 5 mL of Middlebrook 7H9 medium with 10% albumin-dextrose-saline (ADS) supplement at 37°C and grown till OD₆₀₀ of 0.4. The cultured bacteria were centrifuged to aspirate out the medium and re-suspended with fresh 7H9 medium. This bacterial solution was incubated with AG for 3 hr and 6 hr time points and 100 μM DHE was added for 1 hr in dark. The suspension was centrifuged to aspirate out any excess compounds and DHE in the medium. The collected bacterial pellet was re-suspended with acetonitrile and the cells were lysed using a probe sonicator for 3 min on ice. The cell lysate was then removed by centrifugation and the supernatant was separated and injected in Agilent high-performance liquid chromatograph (HPLC) attached with a fluorescence detector (excitation at 356 nm; emission at 590 nm) for analysis. Zorbax SB C-18 reversed-phase column (250 × 4.6 mm, 5 μm) was used and water: acetonitrile (0.1% trifluoroacetic acid) was applied as mobile phase while flow rate was maintained at 0.5 ml/min. The HPLC method used was as described previously (Kalyanaraman et al., 2014 (link)).

To determine the vitamin C content, 0.5 g of the sample was weighed and extracted with 2 mL of 1% metaphosphoric acid using ultrasonic water bath (15 min). Then 1 mL of metaphosphoric acid was added to the sample and it was placed in the bath again. After 15 minutes, 5 mL of metaphosphoric acid was added. Then, the sample was centrifuged. Thero1,4-dimercapto-2,3-butandiol (0.2 mL) was added to the supernatant (0.2 mL) and then diluted with water to 2 mL. The content of vitamin C was measured with a high-performance liquid chromatograph (Agilent Technology, Santa Clara, CA, USA) with an Infinity Bin Pump DAD 1290 detector and a Luna Phenomenex column (4.6 × 250 mm, 5 μm) (Torrance, CA, USA). A mixture of potassium dihydrogen phosphate (pH = 5.0) and methanol was the mobile phase (0.05 mol/L KH₂PO₄:methanol, 97:3). The gradient of the potassium dihydrogenphosphate mixture ranged from 95% at the first min. up to 78% at the sixth min. The content of ascorbic acid was measured at a wavelength of 245 nm using the previously prepared standard curve [62 ].

Chromatographic analyses were performed using a high performance liquid chromatograph (Agilent Technologies Inc., Santa Clara, CA, USA) coupled to an electrospray ionization (ESI) mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). Chromatographic separation was conducted on an Agilent Zorbax SB-C18 column (3.0 mm × 150 mm, 3.5 μm). The mobile phases consisted of 0.1% formic acid–water (A) and acetonitrile (B), and the gradient elution program was set as follows: 0 min, 5% B; 5–10 min, 11% B; 15 min, 19% B; 20–24 min, 24% B; 25 min, 27% B; 28 min, 35% B; 30 min, 38% B and 35 min, 70% B. The flow rate was 0.8 mL·min⁻¹, injection volume was 15 μL, column temperature was 30 °C and detection wavelength was 270 nm. The ESI was applied in negative ion modes for mass analysis and detection. The optimized parameters were as follows: capillary voltage, 3000 V; conical-hole voltage, 60 V; nebulizing-gas pressure, 35 psi; drying-gas flow rate, 10 L·min⁻¹; drying-gas temperature, 320 °C; and mass spectral range, m/z 100–2000.

High-performance liquid chromatograph (Agilent Technologies, Waldbronn, Germany); FlavourSpec^® Flavour Analyzer (G.A.S. It is based on gas chromatography ion mobility spectrometry (GC-IMS), which has both the high separation of gas chromatography and the high sensitivity of ion mobility spectrometry, and can detect trace volatile organic compounds in the samples without enrichment and concentration and other pre-processing to maintain the original flavor of the flavor samples, which is very suitable for the analysis of aroma components. The accompanying software can generate the sample aroma fingerprints, which can easily realize the comparison of sample differences and consistency control); CJJ-931 dual-magnetic heating stirrer (Jiangsu Jintan Jincheng Guosheng Experimental Instrument Factory, Jiangsu); hgs-12 electric thermostatic water bath, KQ-300E ultrasonic cleaner snowflake ice machine (Beijing Changliu Scientific Instrument Co., Ltd. Beijing, China); FA1004B electronic balance (Shanghai Yue Ping Scientific Instrument Co., Ltd. Shanghai, China); IMark enzyme labeling instrument (Biorad, Philadelphia, PA, USA); high-speed freezing centrifuge (Allegra 64R, USA); −80 °C ultra-low-temperature refrigerator (Beijing Chengmaoxing Science and Technology Development Co., Ltd. Beijing, China); WAX columns (RESTEK, Bellefonte, PA, USA).

Using 1% soybean protein isolates as a substrate, the ability of HypZn to hydrolyze natural protein was studied: 10 U recombinant HypZn was added to a 10-mL reaction system, 0.1 g soybean protein isolates was hydrolyzed for 3 h under the optimal conditions, and the degree of hydrolysis was determined by the OPA method [24 ]. Trypsin, pepsin, papain, acid protease and alkaline protease were used as controls.
The molecular weight and distribution of the hydrolysates were analyzed on a TSKGEL G2000 SWXL column. The chromatographic conditions were as follows: mobile phase, 0.05 M phosphate buffer (pH 7.0) was added to a 0.3 M NaCl; column temperature, 30°C; flow rate, 0.5 mL/min; DAD detector, UV 220 nm; and injection volume, 10 μl. HPLC analysis was performed on an Agilent high-performance liquid chromatograph.