Modification of fast-slow differential agar medium for selective isolation of potential starter lactic acid bacteria from cheese

Starter Lactic Acid Bacteria (LAB) are responsible for converting lactose to lactic acid during cheese manufacturing and, as a result, play a critical role in defining the attributes of the final product. There is great interest in isolating novel starter LAB strains to provide alternatives to existing industry cultures or to help enhance the quality and safety of cheeses traditionally made without starter cultures addition [1]. The Fast-Slow Differential Agar (FSDA) medium was developed in 1984 and still remains the standard to rapidly differentiate fast and slow milk-coagulating lactic streptococci and thus avoid screening a large number of isolates for acid production capacity [2]. However, we found that FSDA was unable to selectively isolate fast acid-producing strains from young, traditional, starter-free Izmir Brined Tulum cheeses, due to the presence of a diverse microbiome including Non-Starter LAB and spoilage Gram-negative microbiota [1, 3]. Here, we describe a modified FSDA (mFSDA) with increased selectivity and recovery efficiency towards lactic streptococci, which was successfully used to rapidly isolate potential starters from Tulum cheeses [1] and could similarly outperform FSDA in raw milk cheeses and other varieties containing high levels of “background” microbiota. The main differences between FSDA and mFSDA media consist in the presence of nalidixic acid, ascorbic acid and yeast extract in mFSDA. These targeted additions provide mFSDA with a two-prong selectivity that (I) suppresses unwanted microbiota, and (II) increases the recovery efficiency of lactic streptocci adept to using milk nutrients. Specifically: • Nalidixic acid is an antibiotic that primarily inhibit Gram-negative bacteria [4]. • Ascorbic acid and yeast extract stimulate the growth of lactic streptococci [5] and were added to complement skim milk in creating an environment favoring the growth of lactose-positive, casein peptides-utilizing LAB. • The pH indicator bromocresol purple enabled the chromogenic discrimination between LAB with different acid production capability.