Authentic compounds for analysis by mass spectrometry, such as hydroxy-3-methoxycinnamic acid (ferulic acid), 4-hydroxy-3-methoxybenzoic acid (vanillic acid), 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 2-methoxy-4-vinylphenol (4-guaiachol) were commercially available. The rice-koji fermentation starter (tane koji) of A. luchuensis was obtained from Ishikawa Tanekoji Ten, Ltd. (Okinawa, Japan). Awamori 101 yeast was purchased from Shinzato-shuzo Co., Ltd. (Okinawa, Japan).
Fermentation and alcohol distillation
Japanese spirit, awamori shochu was produced by the general procedures as follows. Briefly, the washed long grain rice (Oryza sativa subsp., indica, 1200 g) was immersed in water for 1 day. It was steamed by an autoclave treatment at 121 °C for 20 min, and it was then cooled to 35 °C. The tane koji (10 g) was next added to the steamed rice and its rice-koji was prepared by incubation at 30 °C for 40 h. To the rice-koji (400 g) was added mother water (720 ml) and yeast (awamori 101 yeast, dry weight 0.1 g), then the fermentation was carried out at 26 °C for 15 days. Following the completion of fermentation, a 50% alcohol, except for the first drops for 5 min, was distillated under decompression. Each analytical sample was collected from the rice-koji suspension, the fermentation products at 5, 10, 15 days and the distilled alcohol. The samples of the rice-koji and fermentation product were centrifuged at 5000 rpm for 30 min (HITACH himac CR22GII, Hitachi Koki, Co., Ltd., Tokyo, Japan). All samples were treated by a solid phase extraction step.
Solid phase extraction
The sample was extracted using a solid phase cartridge column (Oasis HLB Cartridge, Waters). Briefly, the column was conditioned with 5 ml of methanol and MQ-water, then the sample (5 ml) was loaded on the column. The column was continuously washed with 10 ml of MQ-water and the sample was collected with 10 ml of methanol. The solvent was evaporated at room temperature (Centrifugal evaporator CVE-310, Unitraput-1000, EYELA) and the residue dissolved in ethanol (1 ml) was used as the analytical sample.
Each sample was measured by LC/MS (Agilent1200, Agilent Technologies) using a photodiode array detector and monitored at 280 nm on a reversed-phase chromatographic column, YMC-Pack Pro C18 (100 × 4.6 mm I.D., 5 μm particle size, YMC Co., Ltd., Japan) at 40.0 °C. The mobile phase consisting of a 5 mM formic acid aqueous solution (10%) and acetonitrile was carried out at the flow rate of 0.8 ml/min by a linear gradient to 50% (10 min) and 100% (5 min) and held for 5 min. The mass spectra were measured under the following conditions: ESI negative ion mode; desolvation temperature, 350 °C; desolvation pressure, 35 psig and desolvation gas flow, 12.01 ml/min (6120 Quadrupole, Agilent Technologies).
The HRESI-mass spectra were measured using an LC/MS/MS (Agilent 6560 IM-QTOF, Agilent Technologies). The LC was then carried out under the same conditions for the LC/MS analysis. The mass spectra were measured under the following conditions: ESI negative ion mode; drying gas temperature, 350 °C; desolvation pressure, 35 psig and desolvation gas flow, 12 ml/min; capillary voltage, 3500 V; nozzle voltage, 500 V; collision, 20 V. Reference ions were used for 19.03632 and 966.000725.
The annotation information of amino acid sequences from A. luchuensis genome (accession nos: BCWF01000001–BCWF01000044, Yamada et al. 2016) was searched using BLASTP against the Swiss-Prot and TrEMBL protein databases from Uniprot (e-value cut-off 1e-5). Candidate genes related to vanillin production were searched based on the keywords, such as vanillin, enoyl (feruloyl), glucosidase and glucosyl transferase.