Malic

Standards of cyanidin (Cy)-, pelargonidin (Pel)- and their 3-O-glucoside (glc), Cy-rutinoside (Cy-rut), Cy-3,5-di-glucoside (Cy-di-glc), Cy-sambubioside (Cy-sam), and Cy-3-O-sophoroside (Cy-sop) were purchased from Polyphenols Laboratory (Sandnes, Norway). Potassium chloride, hydrochloric acid, methanol, acetonitrile, acetone, Folin reagent, phosphoric and trifluoroacetic acid (TFA) were from Merck (Darmstadt, Germany). Ascorbic acid, 4-dimethylamino-cinnamaldehyde (DMAC), ammonium acetate, acetic acid, glucose, fructose, saccharose, citric, isocitric, succinic, malic, fumaric, oxalic, tartaric, cis-aconitic and quinic acid were provided by Sigma-Aldrich (St. Louis, MO, USA). Extrasynthese (Genay, France) supplied catechin (CAT), epicatechin (EC), procyanidin C1 (PC1), procyanidin A2 (PA2) and punicalagin. Water was obtained from the Arium pro apparatus (Sartorius, Milan, Italy). The freeze-dried raspberries (Rubus idaeus) were a gift from FutureCeuticals (Momence, IL, USA).

Organic acids were determined based on protocols described by Sánchez-Mata et al. [46 (link)]. Extraction was performed with 0.5 g of sample in 25 mL of 3% m-phosphoric acid and analyzed using an HPLC-UV methodology. The HPLC equipment used was a liquid chromatograph (Micron Analítica, Madrid, Spain) equipped with a Sphereclone ODS (2) 250 * 4.60 mm, 5 µm Phenomenex column, isocratic pump (model PU-II), an AS-1555 automatic injector (Jasco, Tokyo, Japan), and a UV-visible detector (Thermo Separation Spectra Series UV100, Waltham, MO, USA), 215 nm for organic acids. The mobile phase was 1.8 mM H₂SO₄ (pH = 2.6), with a flow rate of 0.4 mL/min for organic acids, and injection volume was 100 µL for samples and serial volumes for the standard curve (20, 30, 40, 50, 60, 70, 80, 90, and 100 µL). The compounds were identified by chromatographic comparisons with authentic standards (quinic (0.152 mg/mL), ascorbic (0.155 mg/mL), malic (0.403 mg/mL), fumaric (0.254 mg/mL) and citric acids (0.307 mg/mL), all from Sigma, St. Louis, MO, USA), using linear calibration curves of all compounds for quantification purposes. All data were analyzed using Biocrom 2000 3.0 software (Biocrom, Madrid, Spain).

Sodium hydroxide solution (0.1 mol/L) was purchased from Sigma-Aldrich (Poznań, Poland). Buffer solutions of pH 2, 4 and 7 were purchased from POCH (Gliwice, Poland). Sucrose, glucose, fructose, tartaric, malic, lactic, acetic, citric and succinic acids were obtained from Sigma-Aldrich (Poznań, Poland), all with a purity level of ≥98%. Analytical standards of cyanidin-3-O-glucoside, peonidin-3-Oglucoside, petunidin-3-O-glucoside, malvidin-3-O-glucoside, delphinidin-3-O-glucoside, myricetin-3-O-glucoside, quercetin-3-O-rutinoside, quercetin-4′-O-glucoside, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside, (+)-catechin, (−)-epicatechin, (−)-epicatechin-3-gallate, procyanidin A1 and A2, trans-resveratrol, and trans-piceid, were purchased from Extrasynthese (Lyon, France). The analytic standards of gallic acid, caftaric acid, protocatechuic acid, coutaric acid, caffeic acid, p-coumaric acid, and ferulic acid were purchased from PhytoLab (Vestenbergsgreuth, Germany). Acetonitrile (HPLC gradient grade) was purchased from POCH (Gliwice, Poland). Formic acid (LC–MS grade) was purchased from Fischer Scientific (Schwerte, Germany).

The strain KPS-11 was inoculated in 100 mL of Pikovskaya’s broth in 500 mL flasks in triplicate and incubated in an orbital shaker at 150 rpm at 28 ± 2°C for up to 10 days (240 h). Twenty micro liter of bacterial culture from each flask was harvested at 5, 7, and 10 days post inoculation, centrifuged at 13,000 × g for 10 min and cell-free supernatant was collected. Phosphate solubilization was determined through Phospho-molybdate blue color method using spectrophotometer (λ = 882 nm) as described by Murphy and Riley (1962) (link). For HPLC analysis, the cell-free supernatant was filtered through 0.2 μm nylon filters (Millipore, USA) and 20 μL was injected to HPLC equipped with Turbochrom software (Perkin Elmer, USA) and C-18 column at a flow rate of 0.6 mL min^-1 using 30:1:70 (v/v/v) methanol: acetic acid: water as mobile phase. Signals were detected at 210 nm. The organic acids gluconic, malic, lactic, oxalic, tartaric, and ascorbic acid (Sigma–Aldrich) were used as standard.

Sugars and organic acids were identified and quantified according to Hernández [15 (link)], with some modifications. Approximately 1 g of sample was diluted in 5 mL of phosphate buffer (pH 7.8), homogenized by Ultra-TurraxTM (IKA L004640, Staufen, Germany) for 1 min, and centrifuged at 15,000× g for 10 min. Finally, samples were filtered through a 0.45 μm Millipore filter. For the determination of the content of sugars and organic acids on samples, an HPLC (high-performance liquid chromatograph) Hewlett-Packard series 1100 (Hewlett-Packard, Wilmington, DE, USA) was used. The elution buffer consisted of 0.1% phosphoric acid with a flow rate of 0.5 mL/min.
Sugars and organic acids were isolated using a Supelco column (Supelcogel TM C-610H column 30 cm × 7.8 mm, Supelco, Inc., Bellefonte, PA, USA) and a precolumn Supelguard (5 cm × 4.6 mm; Supelco), and the absorbance was measured at 210 nm using a diode-array detector (DAD). Standards of sugars (glucose, fructose, sucrose, raffinose, maltitol, and sorbitol) and organic acids (oxalic, citric, tartaric, malic, quinic, shikimic, succinic and fumaric) were obtained from Sigma (Poole, UK). Calibration curves were used for the quantification of sugars and organic acids, showing good linearity (R² = 0.999). Results for both organic acids and sugars were expressed as concentrations g/L of dry weight (dw).

Acetonitrile, formic acid, methanol, DPPH (1,1-diphenyl-2 picrylhydrazyl radical), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), TPTZ (2,4,6-tri(2-pyridyl)-s-triazine), acetic acid, phloroglucinol, arbutin, caffeic acid, betulinic, oleanolic and ursolic acid, fructose, glucose, sorbitol and sucrose, and malic, oxalic, citric, shikimic, succinic, and fumaric acids were purchased from Sigma-Aldrich (Steinheim, Germany). Quercetin 3-O-glucoside, kaempferol 3-O-glucoside, cyanidin 3-O-glucoside, isorhamnetin 3-O-glucoside, apigenin 7-O-glucoside, chlorogenic, quinic, p-coumaric and ferulic acids, (+)-catechin, and procyanidin B2 were purchased from Extrasynthese (Lyon, France). Sodium hydroxide (CAS 1310-73-2), iodine (CAS 7553-56-2), and potassium iodide (CAS 7681-11-0) were purchased from CHEMPUR (Piekary Slaskie, Poland).