J w db wax capillary column

The volatile composition of the YD powders was characterized by SPME–GC–MS, as reported previously (Comuzzo et al. 2015b (link)). Analyses were carried out using a GC-17A gas chromatograph equipped with a QP-5000 mass spectrometer (Shimadzu, Kyoto, Japan). Autolysate samples (2.00 g) were introduced in 50 mL amber glass vials sealed with PTFE/silicone septa. Vials were pre-conditioned for 15 min at 40 °C before microextraction, and SPME was run at the same temperature for 15 min, using a 2 cm 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane fiber (Supelco, Bellefonte, PA, USA). A J&W DB-Wax capillary column, 30 m × 0.25 mm i.d., 0.25 µm film thickness (Agilent Technologies Inc., Santa Clara, CA, USA) was used for the GC separation, with the following operating conditions: 40 °C for 1 min, then 4 °C min⁻¹, up to 240 °C, with a final holding of time of 15 min. Injection was performed in splitless mode with 60 s of splitless time; injection port and transfer line were set at 250 and 240 °C respectively. Carrier gas was helium, at a linear flow rate of 35 cm s⁻¹.

Sake meter value, sake acidity, and amino acid levels were measured as previously described⁶ . Organic acids were quantified using HPLC with a Shim-pack SCR-102H column (Shimadzu, Kyoto, Japan). Aroma components were quantified using headspace GC with a J & W DB-WAX capillary column (30 m × 0.32 mm internal diameter × 0.50 μm film thickness; Agilent Technologies, Santa Clara, California, USA). Ethanol concentration was measured using an ethanol analyser (RIKEN KEIKI, Tokyo, Japan). Sugars were determined by monitoring post-column derivative reducing sugars separated using a Prominence reducing-sugar HPLC analytical system (Shimadzu) equipped with a fluorescence detector. Sake fermented with MC87-46 and K901 was separated on a Shim-pack 4.0 × 250-mm ISA-07/S2504 column (Shimadzu) with a linear gradient of 0.1 M potassium borate buffer (pH 8.0) and 0.4 M potassium borate buffer (pH 9.0) for 140 min at a flow rate of 0.6 mL/min^{37 (link),38 (link)}. Sugars were also lyophilized, trimethylsilylated, and analysed using a GCMS-QP2010 (Shimadzu) equipped with a J & W DB-5MS capillary column (30 m × 0.25 mm internal diameter × 0.25 μm film thickness; Agilent Technologies)^{39 (link)}.

FAs were extracted from several mg of lyophilized powder as described by Guo et al. (2019b (link)). FA methyl esters were analyzed with an Agilent GC–MS device (5973 inert mass spectrometer combined with 6890N gas chromatograph) equipped with an Agilent J&W DB-WAX capillary column (30 m × 0.25 mm × 0.25 µm) in a splitless mode. The temperature was set at 150°C for 3 min, then increased to 240°C at 10°C min⁻¹, and kept at 240°C for 5 min. For PA analysis, 5–10 mg of lyophilized powder was treated with 0.01% (w/v) butylated hydroxytoluene in isopropanol (100 µL mg⁻¹) at 75°C for 18 min to deactivate lipid-hydrolyzing enzymes, and extracted with chloroform:methanol:300 mM ammonium acetate (30:41.5:3.5; 300 µL mg⁻¹) at 4°C for 30 min. The samples were clarified by centrifugation at 12,000 rpm, 4°C for 5 min. After another round of extraction, the supernatants were combined and dried under N₂ gas. The extracted lipids were subject to normal phase analysis at LipidALL Technologies Company Ltd. (Changzhou, China) using an Exion UPLC-QTRAP 6500 Plus (Sciex) LC–MS/MS equipped with a Phenomenex Luna 3 µm silica column (internal diameter 150 × 2 mm) as reported previously (Liu et al., 2020 (link)). Individual PA species were quantified by reference to the spiked internal standards d7-PA33:1(15:0/18:1) and DMPA.