Butanal

Commercially available food additive products were used in the human sensory test. Food-grade NaCl was purchased from Naikaisyoji (Tokyo, Japan), and monosodium glutamate (MSG) was purchased from Ajinomoto Co., Inc. (Tokyo, Japan). All flavor compounds (of food additive grade), including propionaldehyde, butanal, isobutylaldehyde, 2-methylbutylaldehyde, pentanal, IVA, hexanal, heptanal, octanal, propionic acid, 2-methylbutyric acid, isovaleric acid, hexanoic acid, heptanoic acid, propyl alcohol, 2-methyl-1-butanol, hexanol, heptanol, methional, methionol, and 3-(methiylthiol) propionic acid, were purchased from Sigma-Aldrich (St. Louis. MO, USA). Deionized water for test solutions and mouth rinsing was supplied by a Mill-Q water purification system (Millipore, Bedford, MA, USA). γ-Glu-Val-Gly was obtained from Ajinomoto Co., Inc. (Tokyo, Japan). NPS-2143, a CaSR inhibitor, was synthesized, as described by Rybczynska et al. [41 (link)]. Chemicals for cell based-assays, such as CaCl₂, ethylenediaminetetraacetic acid (EDTA), and probenecid, were purchased from Sigma-Aldrich (St. Louis. MO, USA).

Sulfuric acid for HPLC analysis was employed (30,743 Honeywell Fluka 95–97%). MEthanol for RP-HPLC was purchased from Merck (34,860, ≥99.9%).
Standard solutions for HPLC (TraceCERT^®, 1,000 mg/L in water) were purchased from Sigma-Aldrich (Milan, Italy). Analyte stock solutions were prepared by dissolving a weighed amount of the pure compound in deionized water and stored at 4°C up to 1 month. Ethanol, ¹³C-labeled Ethanol, acetoin, acetone, butanal, 2-methylbutanal, 3-methylbutanal, butanedione, butanol, 2-methyl-butanol, 3-methyl-butanol, butanone, hexanal, mEthanol, methylacetate, 2-pentanol, propanal, 2-methylpropanal, propanol, 2-methyl-propanol, and sec-butanol were purchased from Sigma-Aldrich (Italy). All chemicals, having purity higher than 99%, were used without any further purification.
Solid-phase microextraction fiber based on 85 um carboxen/polydimethylsiloxane (CAR/PDMS) was employed for the preconcentration of volatile compounds in the HS.
Helium 5.6 IP was purchased from SOL Group Spa (Italy) and was further purified with a super clean filter purchased from Agilent Technologies (United States) to remove water, oxygen, and hydrocarbon contaminants.
Preparation/dilution of samples and solutions was performed gravimetrically using ultrapure water (Milli-Q; 18.2 MΩ cm⁻¹ at 25°C, Millipore, Bedford, MA, United States).

The activity of recombinant WS11-AldH and AD45-AldH was assayed spectroscopically by following the change in absorbance at 340 nm associated with the formation of NAD(P)H from NAD(P)⁺. The 1-mL reaction mixture, prepared in a quartz cuvette, contained 50 mM sodium carbonate buffer (pH 10.0), 0.1 mg WS11-AldH or AD45-AldH, and 5 mM substrate (acetaldehyde [Sigma; W200379], propanal [Thermo Fisher; 131510250], butanal [Sigma; 8.01555], 2-methyl butanal [Sigma; M33476], tiglic aldehyde [Sigma; 192619], and pentanal [Sigma; 110132]). All chiral aldehydes purchased for use in this study were chemically synthesized and therefore were racemic mixtures. The reaction was initiated by the addition of 5 mM NAD⁺/NADP⁺, and the cuvette was mixed by inversion. The change in absorbance at 340 was measured for 5 min.
WS11-AldH and AD45-AldH were assayed at substrate concentrations in the range of 10 mM to 25 µM for the three fastest substrates, butanal, 2-methylbutanal, and propanal. Michaelis-Menten parameters were calculated using GraphPad Prism 8.0.1. For 2-methylbutanal, the substrate inhibition model was used at the high concentrations, as the substrate began to inhibit activity.
WS11-IsoH dehydrogenase assays (described above), using 1 mM HGMB, were performed with the addition of 0.1 mg/mL WS11-AldH or AD45-AldH to determine if the rate of the dehydrogenase activity increases.

Dansyl hydrazine (DH), glyoxal, crotonaldehyde, 2,4-nonadienal (NDE), 2,4-decadienal (DDE), heptadecanal, hexadecanal, irsogladine maleate, octadecanal, pentadecanal, rebamipide, teprenone, tetradecanal and troxipide were purchased from Tokyo Chemical Industry (Tokyo, Japan). The NF-κB inhibitor SM-7368 was obtained from Merck KGaA (Darmstadt, Germany). p-Toluenesulfonic acid (p-TsOH) and the RCs including propanal, pentanal, butanal, 2-hexenal, hexanal, 2-heptenal, heptanal, octanal, 2-nonenal, nonanal, decanal, undecanal, dodecanal and tridecanal were obtained from Sigma-Aldrich (St. Louis, MO, USA). 4-Hydroxy-2-hexenal (HHE), 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE) and 4,5-epoxy-2-decenal (EDE) were purchased from the Cayman Chemical Company (Ann Arbor, MI, USA). Ecabet sodium hydrate, p-benzyloxybenzaldehyde (p-BOBA), sofalcone and other chemicals used in the current study were purchased from Wako Pure Chemical Industries (Osaka, Japan). 8-Heptadecenal (8-HpDE), 8,11-heptadecadienal (8,11-HpDDE) and 8,11,14-heptadecatrienal (8,11,14-HpDTE) were synthesized by a method previously described [26 (link), 27 (link)]. Secosterol-A and -B were synthesized according to the procedure reported by Wentworth et al. [28 (link)]. Stock solutions of the RCs and an internal standard (IS) (p-BOBA, 10 mM) were prepared separately in acetonitrile and stored at −20°C prior to use.

Eight commercial samples for each type of vinegar were randomly purchased from a local market. The product information was extracted from the labels, and this is outlined in Table S1. Organic acid standards (formic acid, acetic acid, pyruvic acid, fumaric acid, oxalic acid, malic acid, pyroglutamic acid, tartaric acid, lactic acid, citric acid, and succinic acid), amino acid standards (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg, Pro, pyroglutamic acid, and GABA), butanal, rutin, gallic acid, catechin, p-coumaric acid, ferulic acid, caffeic acid, chlorogenic acid, and 2-octanol were obtained from Sigma-Aldrich (St. Louis, MO, USA). ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic), FRAP (ferric reducing/antioxidant power), DPPH (1,1-Diphenyl-2-picrylhydrazyl), K₂S₂O₈, FeCl₃, NaCl, Na₂CO₃, NaNO₂, Al(NO₃)₃, NaOH, H₂O_2, NH₃.H₂O, 2,4,6-tripyridyl-s-triazine, phosphate, soybean lecithin_, deoxyribose, trichloroacetic acid, fulin, methanol, and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).