2.2.2. Preparation of Starter Cultures and Fermentation of Milk. The LAB strains to be used as inocula were prepared by
transferring a loopful of an overnight culture fromMRS agar into 10 mL MRS broth and incubated at 35∘C for 24 h. One
hundred microliters of the 24 h old culture was transferred into 10 mL MRS broth and incubated at 35∘C for 16h (overnight). Subsequently, cells were harvested by centrifugation at 5000 g for 10 min (4∘C); washed three times with 20 mL sterile diluent (Merck), pH 7.2 ± 0.2; and finally suspended in 10 mL of sterile diluent, and these served as the isolate
inocula. Flasks containing 500 mL of fresh milk pasteurized at 60∘C for 30 min in a water bath were inoculated in duplicate
at 35∘C for 24 h. Single and combined starter cultures used for the fermentation of milk to produce Nunu are shown in Table 1.