Scl 10avp

SGT and LCGT eluates were measured by HPLC as described previously [11 (link)]. In brief, catechins and caffeine in the eluates were measured by HPLC (SCL-10Avp, Shimadzu, Kyoto, Japan; Develosil packed column ODS-HG-5, 150 × 4.6 mm, Nomura Chemical Co. Ltd., Seto, Aichi, Japan) according to the method of Horie et al. [22 (link)]. Catechins and caffeine were measured at 280 nm. Free amino acids in tea leaves were measured by HPLC as described above using homoserine as the internal standard [23 (link)]. Amino acids were detected at an excitation wavelength of 340 nm and at an emission wavelength of 450 nm using an RF-535 UV detector (Shimadzu, Japan). The relative standard deviation (RSD%) of precision and repeatability were <5.0%. The recoveries of catechins, caffeine, and free amino acids were 99 ± 4%, 98 ± 4%, and 98 ± 3%, respectively.

The molecular weight distribution of DES-lignin was analyzed by gel permeation chromatography on the HPLC system (Shimadzu HPLC, Kyoto, Japan), which comprises the following parts: a system controller SCL-10AVP, an on-line degasser DGU-14A, a low-pressure gradient valve FCV-10ALVP, an HPLC pump LC-10ATVP, an autosampler SIL-20AHT, and a column oven CTO-10ACVP. The system was equipped with a sequentially connected guard column (50 mm × 7.8 mm) and two Jordi Gel DVB 500A (300 mm × 7.8 mm) columns in a series. Separations were run by using THF with 1% acetic acid as an eluent with a constant flow rate of 0.8 mL min ${}^{-1}$ . The detector parameters were as follows: an HPLC nebulizer; 40 ${}^{°}$ C; an air pressure of 3.5 bar; a gain of 3; no-split mode. The injection volume of the autosampler was 50 $\mathsf{\mu }$ L. The oven column was set to 40 ${}^{°}$ C. Polystyrene Standards (Perkin-Elmer, Norwalk, CT, USA) were used to calibrate the columns. Chromatogram evaluation was implemented by commercial software for GPC analysis CLASS-VP Version 1.03 (Shimadzu).

The total carotenoids in the RYCs were first extracted by breaking the yeast cells using the modified method of Manowattana et al. [23 (link)]. The RYC pellet was harvested from 10 mL of the culture broth by centrifugation at 4430× g and 4 °C for 10 min. The RYC pellet was washed twice with n-hexane and once with distilled water and extracted in a screw-capped tube containing 10 mL of acetone and 4 g of glass beads in the presence of 100 ppm ascorbic acid by vortexing for 15 min. The broken RYCs were centrifuged at 4430× g and 4 °C for 10 min, the clear supernatant collected and flushed with N₂ gas to ensure complete drying.
Quantitative analysis of the carotenoids was carried out according to Chaiyaso and Manowattana [22 (link)]. In brief, the extracted carotenoids were redissolved in 1.0 mL of n-hexane, filtered through a 0.2 μm nylon membrane (ALWSCI, Shaoxing, Zhejiang, China) and subjected to high-performance liquid chromatography (HPLC; SCL-10Avp, Shimadzu, Kyoto, Japan) equipped with a C18 column (4.6 × 250 mm; 5 μm, Restek, Bellefonte, PA, USA). The mobile phase consisted of acetonitrile: dichloromethane: methanol (80:10:10, v/v/v), and the flow rate was 1.0 mL/min at 30 °C. The total carotenoid content was detected using a UV–VIS detector (SPD-10Avp, Shimadzu, Kyoto, Japan) operating at 454 nm for 45 min.