2.7.4. Inhibition of extracellular lipase: plate assay method

Using the same method as in the qualitative assay for lipase detection (section 2.7.1), the most active anti-M. globosa lipase activity fraction (CMPE1) was also tested. At a final volume of 1 mL, an equal volume of CMPE1 in 2× MLN broth (concentrations 0.06–1.00 mg/mL) and yeast cell suspension (density of ∼10⁷ cells/mL) were mixed and incubated at 30 °C for 24 h and 48 h with shaking at 200 rpm.

After reaching the indicated incubated time points, the yeast cells from each treatment were collected by centrifugation at 15,322×g (12,000 rpm), room temperature for 5 min. They were then washed twice with PBS, adjusted in cell density to obtain cell suspensions (50 μL) in each well of TW60-Vic B agar of ∼10⁶ cells/mL final density. The agar medium plates were then incubated at 30 °C for 1–2 days before observing the results as previously described in section 2.7.1.