Vanillin

The total phenolic content was determined by employing the methods given in the literature (Slinkard and Singleton, 1977 (link)) with some modification. Sample solution (1 mg/mL; 0.25 mL) was mixed with diluted Folin–Ciocalteu reagent (1 mL, 1:9, v/v) and shaken vigorously. After 3 min, Na₂CO₃ solution (0.75 mL, 1%) was added and the sample absorbance was read at 760 nm after a 2 h incubation at room temperature. The total phenolic content was expressed as milligrams of gallic acid equivalents (mg GAE/g extract) (Vlase et al., 2014 ).
The total flavonoids content was determined using AlCl₃ method (Zengin et al., 2014 (link)). Briefly, sample solution (1 mg/mL; 1 mL) was mixed with the same volume of aluminum trichloride (2%) in methanol. Similarly, a blank was prepared by adding sample solution (1 mL) to methanol (1 mL) without AlCl₃. The sample and blank absorbances were read at 415 nm after a 10 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. Rutin was used as a reference standard and the total flavonoid content was expressed as milligrams of rutin equivalents (mg RE/g extract) (Mocan et al., 2015 (link)).
The total saponins content of the extract was determined by the vanillin-sulfuric acid method (Aktumsek et al., 2013 (link)). Sample solution (1 mg/mL; 0.25 mL) was mixed with vanillin (0.25 mL, 8%) and sulfuric acid (2 mL, 72%). The mixture was incubated for 10 min at 60°C. Then the mixture was cooled for another 15 min, followed by the sample absorbance measurement at 538 nm. The total saponin content was expressed as milligrams of quillaja equivalents (mg QAE/g extract).
The total triterpenoids content of the extracts was determined according to Zhang et al. (2010) (link) method with some modifications. Briefly, sample solution (1 mg/mL; 500 μL) was mixed with the vanillin–glacial acetic acid (5%, w/v, 0.5 mL) and 1 mL of perchloric acid. The mixture was incubated at 60°C for 10 min, cooled in an ice water bath for 15 min and then 5 mL glacial acetic acid was added and mixed well. After 6 min, the absorbance was read at 538 nm. Oleanolic acid was used as a reference standard and the content of total triterpenoids was expressed as oleanolic acid equivalents (mg OAE/g extract) through a calibration curve with oleanolic acid.
HPLC-PDA analyses were performed on a Waters liquid chromatograph equipped with a model 600 solvent pump and a 2996 photodiode array detector, and Empower v.2 Software (Waters Spa, Milford, MA, United States) was used for acquisition of data. A C18 reversed-phase packing column (Prodigy ODS (3), 4.6 × 150 mm, 5 μm; Phemomenex, Torrance, CA, United States) was used for the separation and the column was thermostated at 30 ± 1°C using a Jetstream2 Plus column oven. The injection volume was 20 μL. The mobile phase was directly on-line degassed by using Biotech DEGASi, mod. Compact (LabService, Anzola dell’Emilia, Italy). Gradient elution was performed using the mobile phase water-acetonitrile (93:7, v/v, 3% acetic acid) (Zengin et al., 2016 (link)). The UV/Vis acquisition wavelength was set in the range of 200–500 nm. The quantitative analyses were achieved at maximum wavelength for each compound.

250 μl of thawed hydrolysate and 15 μl of sorbitol (0.1000 g/100 ml aqueous) were transferred to a vial and concentrated to dryness under a stream of N₂. The internal standard was added to correct for subsequent differences in derivatization efficiency and changes in sample volume during heating. Dried extracts were dissolved in 500 μl of silylation–grade acetonitrile followed by the addition of 500 μl N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) (Thermo Scientific, Bellefonte, PA), and samples then heated for 1 h at 70°C to generate trimethylsilyl (TMS) derivatives [43 (link)]. After 1 day, 1-μl aliquots were injected into an Agilent Technologies Inc. (Santa Clara, CA) 5975C inert XL gas chromatograph-mass spectrometer, fitted with an Rtx-5MS with Integra-guard (5% diphenyl/95% dimethyl polysiloxane) 30 m × 250 μm × 0.25 μm film thickness capillary column. The standard quadrupole GCMS was operated in the electron ionization (EI) (70 eV) mode, with 6 full-spectrum (50–650 Da) scans per second. Gas (helium) flow was 1.33 ml per minute with the injection port configured in the splitless mode. The injection port, MS Source, and MS Quad temperatures were 250°C, 230°C, and 150°C, respectively. The initial oven temperature was held at 50°C for 2 min and was programmed to increase at 20°C per min to 325°C and held for another 11 min, before cycling back to the initial conditions. A large user-created database (>1600 spectra) of mass spectral EI fragmentation patterns of TMS-derivatized compounds, as well as the Wiley Registry 8th Edition combined with NIST 05 mass spectral database, were used to identify the metabolites of interest to be quantified. Peaks were reintegrated and reanalyzed using a key selected ion, characteristic m/z fragment, rather than the total ion chromatogram, to minimize integrating co-eluting metabolites. The extracted peaks of known metabolites were scaled back up to the total ion current using predetermined scaling factors. Unidentified metabolites used the scaling factor for the internal standard (sorbitol) and were denoted by their RT as well as key m/z fragments. The mass-to-charge ratios used as extracted ions were as follows: iso-sinapyl alcohol (354), iso-sinapic acid (368), iso-syringin (354), 5-hydroxyconiferyl alcohol-4-O-glucoside (412), 5-hydroxyconiferyl alcohol-4-O-glucoside (412), 3,4-dihydroxybenzoic acid (370), xanthine (368), hypoxanthine (265), succinic acid (247), guanosine (324), uracil (241), citraconic acid (259), guanine (352), 5-hydroxyferulic acid (411), uridine (258), maleic acid (245), secoisolariciresinol (560), 5-oxo-proline (156), adenine (264), 1-O-trans-feruloylglycerol (249), vanillin (297, 194), ferulic acid (338), adenosine (236), p-coumaric acid (308), caffeic acid (396), p-hydroxybenzaldehyde (392, 194), coniferyl alcohol (324), 5-hydroxyconiferyl alcohol (412), coniferyl aldehyde (323), guaiacylglycerol (297), sinapyl aldehyde (353), syringylglycerol (327), p-hydroxyphenylpyruvic acid (396), syringaresinol (327), pinoresinol (502), hydroxymethylfurfural (183). Peaks were quantified by area integration and the concentrations were normalized to the quantity of the internal standard recovered, volume of sample extracted, derivatized, and injected.

In this study, most of the chemicals, reagents, and standards were analytical grade and purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Gallic acid, L-ascorbic acid, vanillin, hexahydrate aluminium chloride, Folin-Ciocalteu’s phenol reagent, sodium phosphate, iron(III) chloride hexahydrate (Fe[III]Cl₃.6H₂O), hydrated sodium acetate, hydrochloric acid, sodium carbonate anhydrous, ammonium molybdate, quercetin, catechin, 2,2′-diphenyl-1-picrylhy-drazyl (DPPH), 2,4,6tripyridyl-s-triazine (TPTZ), and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from the Sigma-Aldrich (Castle Hill, NSW, Australia) for the estimation of polyphenols and antioxidant potential. Sulfuric acid (H₂SO₄) with 98% purity was purchased from RCI Labscan (Rongmuang, Thailand). HPLC standards including gallic acid, p-hydroxybenzoic acid, caftaric acid, caffeic acid, protocatechuic acid, sinapinic acid, chlorogenic acid, syringic acid, ferulic acid, coumaric acid, catechin, quercetin, quercetin-3-galactoside, diosmin, quercetin-3-glucuronide, epicatechin gallate, quercetin-3-glucoside, kaempferol and kaempferol-3-glucoside were produced by Sigma-Aldrich (Castle Hill, NSW, Australia) for quantification proposes. HPLC and LC-MS grade reagents including methanol, ethanol, acetonitrile, formic acid, and glacial acetic acid were purchased from Thermo Fisher Scientific Inc. (Scoresby, VIC, Australia). To perform various in vitro bioactivities and antioxidant assays, 96 well-plates were bought from the Thermo Fisher Scientific (VIC, Australia). Additionally, HPLC vials (1 mL) were procured from the Agilent technologies (VIC, Australia).

LB medium was used for Escherichia coli, CM2B medium (10 g/L polypeptone, 10 g/L yeast extract, 5 g/L NaCl, 10 μg/L biotin, pH 7.0 adjusted with KOH) for C. glutamicum, and YPD medium (10 g/L yeast extract, 20 g/L Bacto peptone, 20 g/L glucose) for Saccharomyces cerevisiae. Culture temperature was set as 30 °C (S. cerevisiae), 31.5 °C (C. glutamicum), or 37 °C (E. coli). A 20 μL aliquot of each glycerol stock of the strains was applied to each agar medium and cultured for 20 h as pre-culture. The obtained cells were washed and suspended in sterile physiological saline. The optical density (OD) at 620 nm (OD_620nm) of the cell suspension was measured, and the cell suspension was inoculated into an L-shaped test tube containing 4 mL medium (initial OD_620nm: 0.02 for “Comparison of vanillin tolerance” and “Identification of the aromatic aldehyde reductase (AAR) that converts vanillin to vanillyl alcohol”, and 0.2 for “Evaluation of vanillin tolerance of FKFC14 strain”). Culturing was performed using a culture apparatus equipped with an automatic OD measurement function (TVS062CA ADVANTEC). OD_660nm was measured every 15 min. For “Comparison of vanillin tolerance”, 0, 1, or 2 g/L vanillin was added to the medium before inoculation. For “Identification of the aromatic aldehyde reductase (AAR) that converts vanillin to vanillyl alcohol”, 1 g/L vanillin was added to the medium before inoculation. For “Evaluation of vanillin tolerance of FKFC14 strain”, 0, 3, or 6 g/L of vanillin was added to the medium in the middle of the log phase. Specific growth rates (μ) were calculated according to the following equation: ln X_t = ln X₀ + μ, where X_t and X₀ are optical density measurements at time t and time 0, respectively.

Vanillin stock solution (1000×) was dissolved in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA), and 0.1% DMSO was included in the working solution to ensure no toxicity to cells. As the vehicle control, 0.1% DMSO was used.

For small-scale production of vanillin, experiments were carried out using 100-ml shake-flasks. 1% fresh overnight culture was inoculated into shake-flasks containing 10 ml SC medium supplemented with 20 μM copper sulfate. The cultures were incubated at 30 °C and 250 rpm for vanillin productions. For gas chromatography-flame ionization detector (GC-FID) analysis of vanillin, 100 μl of supernatant was extraction with 900 μl ethyl acetate before subjected to GC-FID analysis. 1 μl of diluted sample was injected into GC-2030 system equipped with an Rtx-5 column (30 m × 250 μm × 0.25 μm thickness). Nitrogen (ultra-purity) was used as carrier gas at a flow rate 1.0 ml min⁻¹. GC oven temperature was initially held at 40 °C for 2 min, increased to 45 °C with a gradient of 5 °C min⁻¹ and held for 4 min. And then it was increased to 230 °C with a gradient 15 °C min⁻¹.
For high-performance liquid chromatography (HPLC) analysis of vanillin, Shimadzu Prominence LC-20A system (Shimadzu, Japan) equipped with a reversed phase C18 column (150 × 4.6 mm, 2.7 μm) and a photodiode array detector was used. The samples were centrifuged and filtered through a 0.2-μm syringe filter before injected to the HPLC system. The mobile phase comprises solvent A (ddH₂O with 0.1% trifluoroacetic acid) and solvent B (acetonitrile with 0.1% trifluoroacetic acid). The following gradient elution was used: 0 min, 95% solvent A + 5% solvent B; 8 min, 20% solvent A + 80% solvent B; 10 min, 80% solvent A + 20% solvent B; 11 min, 95% solvent A + 5% solvent B. The flow rate was set at 1 ml min⁻¹. The levels of vanillin and other aromatic compounds were monitored at the absorbance of 275 nm.