# Microbiota and Metabolite Profiling of Spoiled Spanish-Style Green Table Olives
## Materials and Methods
### Chemical Analyses  
The pH, titratable acidity, combined acidity, sodium chloride, and total polyphenols were measured following the routine procedures used in our laboratories [10]. Lactic acid, succinic acid, and ethanol were analyzed by HPLC using a C18 column and deionized water (pH adjusted to 2.2 using concentrated H\({}_{3}\)PO\({}_{4}\)) as the mobile phase and a refractive index detector [39]. Volatile compounds, except volatile fatty acids, were analyzed by HS-SPME-GC-MS following the procedure described in a previous work [14] with modifications. Brine samples (\(\approx\)50 mL) were neutralized to pH 7-8 with magnesium oxide and the aid of a pH meter and magnetic stirrer. The precipitate was separated by filtration and an aliquot of filtrate (2.5 mL) was inserted into a 15 mL glass vial. After the addition of 7.5 mL of a NaCl solution (300 g L\({}^{-1}\)) and 100 \(\upmu\)L of 3-octanol (2 mg L\({}^{-1}\)), the vial was closed and placed in a water bath adjusted to 40 \({}^{\circ}\)C. The vial was equilibrated for 15 min at 40 \({}^{\circ}\)C and stirred at 600 rpm using a stirring bar. The headspace volatile compounds were extracted for 30 min on a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber (1 cm, 50/30 \(\upmu\)m; Supelco, Bellefonte, PA, USA). The volatile compounds adsorbed on the SPME fiber were desorbed at 265 \({}^{\circ}\)C for 15 min in the injector port of a GC interfaced with a mass detector (internal ionization source: 70 eV) with a scan range from \(m/z\) 30 to 400 (GC model 7890A and mass detector model 5975C, Agilent Technologies, Santa Clara, CA, USA). The chromatographic conditions along with the identification and quantification procedures are described in [14]. Three replicates per each sample were prepared and analyzed. Volatile fatty acids were analyzed by HS-SPME-GC-MS using a new procedure. An aliquot (2.5 mL) of brine, 0.2 mL of 2 N HCl, and 7.3 mL of a NaCl solution (300 g L\({}^{-1}\)) were placed in a 15 mL vial. As the internal standard, 20 \(\upmu\)L of 2-ethylbutyric acid (900 mg L\({}^{-1}\)) were used. After equilibration for 15 min at 40 \({}^{\circ}\)C, a polyacrylate-coated SPME fiber (PA, 85 \(\upmu\)m; Supelco) was exposed to the sample HS for 30 min at 40 \({}^{\circ}\)C. Volatile acids were desorbed at the GC injection port at 250 \({}^{\circ}\)C for 15 min. The GC-MS system was equipped with a DB-FFAP column (30 m \(\times\) 0.25 mm i.d., 0.25 \(\upmu\)m; J&W Scientific, Folsom, CA, USA). The helium flow rate was 1 mL/min. The column was maintained at 80 \({}^{\circ}\)C for 2 min, ramped at 8 \({}^{\circ}\)C/min to 200 \({}^{\circ}\)C, and held for 15 min. The transfer line temperature was maintained at 230 \({}^{\circ}\)C. For the mass detector conditions, the quadrupole and ion source temperatures were maintained at 150 and 230 \({}^{\circ}\)C, respectively. The concentrations of the volatile fatty acids were calculated from calibration curves with standards of acetic, propionic, isobutyric, butyric, 3-methylbutyric, 2-ethylbutyric (IS), valeric, 4-methylvaleric, caproic, heptanoic, and cyclohexanoic acids and expressed in mg L\({}^{-1}\). The following equation was used:  
\[\left[\mathrm{x}\right]_{\mathrm{sample}}=\mathrm{peak\ area\ (x)}\times \frac{\left[\mathrm{IS}\right]}{\mathrm{peak\ area\ (IS)}}\times\frac{\mathrm{slope\ (IS)}}{\mathrm{slope\ (x)}}\]  
where \(\left[\mathrm{x}\right]_{\mathrm{sample}}\) is the concentration of the compound in the brine sample, [IS] is the concentration of the internal standard (IS), and slope (IS) and slope (x) are slopes from the calibration curves of the internal standard and compound x, respectively. The calibration curves gave coefficients of determination (\(R^{2}\)) ranging from 0.988 to 0.999 and detection limits from 0.02 to 7 mg L\({}^{-1}\). The relative standard deviation (RSD) ranged from 2 to 11%, and recovery rates from 91 to 111%. Each sample was analyzed in triplicate.