Ri 4030

PPL activity in hexane was determined by measuring the esterification reaction of ibuprofen by the enzyme using HPLC. PPL (75 mg), ibuprofen (0.20 M), and sorbitol (0.16 M) were equilibrated to a water activity of 1 using atmospheric equilibration over water-saturated potassium sulfate [107 (link)]. The mixture was incubated at 37 °C on a shaking water bath (300 rpm). Samples were withdrawn during the reaction for quantification of the decrease of ibuprofen by HPLC (JASCO HPLC with C18 reverse phase Synergi 4 µm Hydro-RP 80 Å − 250 × 4.6 mm equipped with refractive index (model RI-4030) and UV–Vis detector (model UV-4070) [108 (link)]. One unit of lipase activity is defined as 1 µmol ibuprofen consumed per min per mg lipase. All measurements were performed at least in triplicate.

Lactose, glucose, galactose, butyl-β-galactoside, acetone, and butanol were determined using an HPLC system (Jasco, Japan), consisting on a refractive index detector RI-4030, a quaternary pump 4180, a column heater CO-4060, an autosampler AS 4050, and interphase LCNETII-ADC. The peaks were integrated using the Chromnav 2.0 software provided by the manufacturer. Samples were eluted through an Aminex^® HPX-87H (300 mm × 7.8 mm) column at a flow rate of 0.4 mL min^–1. Mobile phase was a mixture of 0.005N sulfuric acid and 0.2% (v/v) of acetonitrile. Column and detector were kept at constant temperatures of 45 and 40°C, respectively. Retention times for lactose, glucose, galactose and butyl-β-galactoside were 10.9, 12.8, 13.7, and 19.5 min, respectively. Acetone, and butanol were quantified employing the same procedure, but using an eluent flow rate of 0.5 mL min^–1. Their retention times were 26.1 and 42.9 min, respectively.

To quantify sugars (glucose, fructose), organic acids (acetic, glucuronic, and gluconic acids), and alcohols (glycerol, ethanol) during kombucha tea trials, HPLC analysis was performed at 0, 7, and 14 days of fermentation using the Extrema LC-4000 system (Jasco, Cremella, Italy) coupled with a refractive index detector RI-4030 (Jasco) set to 35 °C. Analytes were separated using a Rezex^TM ROA-Organic Acid H + (8%) column (300 × 7.8 mm; Phenomenex, Castel Maggiore, Italy) equipped with a Carbo-H (4 × 3.0 mm) guard column (Phenomenex). The column was maintained at a constant temperature of 80 °C and under an isocratic mobile phase consisting of 5 mM H₂SO₄ (Honeywell, Rodano, Italy) at a flow rate of 0.8 mL/min. Before analysis, samples were centrifugated at 6000× g for 5 min, filtered with 0.22 µm syringe filters SPHEROS (LLG Labware, Meckenheim, Germany), and appropriately diluted with 5 mM H₂SO₄. Calibration curves were prepared in a range from 0.05 to 6 g/L.

The chemical composition (cellulose, lignin, emicellulose, moisture and ash) of untreated rachis sample and of all the residual solid fraction post pretreatments were determined following standard Technical Association of Pulp and Paper Industry (TAPPI) protocols [41 ]. The monosaccharides (glucose, xylose and arabinose) released after hydrolysis were quantitatively estimated using High Performance Liquid Chromatography (HPLC) with a refractive index detector (Jasco RI-4030, Easton, MD, USA). A Rezex ROA-Organic Acid H⁺ (8%), 300 × 7.8 mm (Phenomenex, Torrance, CA, USA) was used at 80 °C. Isocratic elution was carried out with H₂SO₄ 0.01M water at 0.6 mL/min. Samples were analyze in triplicate. Infrared spectroscopic analysis of residual lignin has been carried out by means of Fourier transform infrared (FT-IR) spectrophotometer (Perkin Elmer Spectrum 1000), using a KBr disc containing 1% finely ground samples. Through the Spectrum 10^® software, supplied with the instrument, the corresponding absorbance spectra were collected within the range of 4400 cm⁻¹ to 500 cm⁻¹, and processed for baseline correction, noise correction (smooth) and identification of the wave numbers of the main peaks.