Fed-batch fermentation

In order to expand the production scale of lipase, a 3 L fermentation was adopted according to the method described¹³. Yeast seed liquid (100 ml) was added to BSM medium (900 ml). The fermentation process consisted of three phases, including the glycerol culture stage, glycerol feeding stage, and methanol induction stage. Glycerol culture was conducted at 30 ℃, with pH 6.0 maintained using NH₄OH. The initial glycerol was depleted for about 20 h and entered the glycerol feeding stage. In the glycerol feeding stage, 50% glycerol (glycerol:water = 1:1, v/v) was added to the fermentation tank until OD₆₀₀ reached 110. After the exhaustion of glycerol in the medium, it entered the methanol induction phase, where the temperature was maintained at 28 ℃, and the concentration of methanol was maintained at 0.1 ± 0.02% (w/v). The fermented samples were centrifuged at 10,000 rpm for 10 min. The cell number, lipase activity, and protein concentration of the supernatant were measured. The concentration of protein was determined using the Bradford method, and bovine serum albumin (BSA) (Beijing Solarbio Science & Technology Co., Ltd.) was used as the standard.