A culture aliquot was inoculated into 5 mL of sterilized orange juice and incubated at 37 C for 15 h. After this time, 0.05 mL was again inoculated into 10mL of sterilized orange juice and incubated at 37 C for 15 h (pre-inoculum) (Pimentel, Madrona, Garcia, & Prudencio, 2015).