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Fed-batch culture was carried out using shaking flasks (Corning, NY, USA) on a rotary shaker at 140 rpm, with an medium volume of 200 ml.
The optimum conditions for the production of glycosylated lactosides were determined (seeded cell number of 5 × 106; 72 h incubation period; lactoside concentration of 100 μM; medium volume of 10.5 ml) with galactosylated lactoside obtained in 11.6% yield.
Two spore cakes (5 mm) were used to inoculate a 250 ml flask having medium volume of 40 ml and then incubated at 28 °C with agitation speed of 160 rpm for 48 h.
We selected the following conditions: seeded cell number of 5 × 106 or 10 × 106 cells; 72 h incubation period; lactoside concentration of 100 or 150 μM; and medium volume of 10.5 ml.
For stirred microcarrier cultures, 100-mL spinner flasks (Bellco Glass, Vineland, USA) were used, with a final medium volume of 80 mL and a stirring speed of 40 rpm.
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