Varian cary 60 uv vis spectrophotometer

The triterpenoid content was determined based on the vanillin-perchloric acid method with slight modifications [48 (link)]. The test solution (200 µL, 1 mg/mL), in a 10 mL tube, was heated to evaporate the solvent and the solid was reconstituted in a vanillin-glacial acetic acid solution (300 µL, 5% w/v) and in perchloric acid (1.0 mL, 70%). The sealed samples were heated for 45 min at 60 °C and afterwards cooled in an ice-water bath followed by the addition of glacial acetic acid (4.5 mL). The absorbance of the sample solutions was measured at 540 nm against a blank using a Varian Cary 60 UV–VIS spectrophotometer (Agilent, Santa Clara, CA, USA). The blank was treated identically with the exception that no vanillin was used. Oleanolic acid (0.0090–0.4000 mg/mL in methanol) was used for calibration and the results were expressed as mg of oleanolic acid equivalents per gram of dry extract (mg OAE/g).

Biomass production was conducted using shake-flask cultivation in 25 mL stoppered conical flasks. The cultivation medium included: 1.5 g L⁻¹ (NH₄)₂SO₄, 0.2 g L⁻¹ MgSO₄·7H₂O, 2.5 g L⁻¹ NaCl, 1.5 g L⁻¹ KH₂PO₄, and 1.5 mL L⁻¹ trace element solution. The concentration of yeast extract and glucose-rich hydrolyzate and the medium pH were varied as per the model design (Table 2). All flasks were incubated at 37 °C in an orbital shaker (New Brunswick Innova 44, Eppendorf) at 200 rpm for the relevant times (Table 2). After incubation, a 1 mL aliquot was aspirated, and cell density was recorded as the optical density at 600 nm (OD₆₀₀) using a spectrophotometer (Varian Cary 60 UV/Vis spectrophotometer; Agilent Technologies) against a blank of the 1 mL respective uninoculated cultivation medium.