Fermented milk products like yogurt have been recognized for their beneficial effects on health. This is largely due to the presence of lactic acid bacteria (LAB) who are responsible for the synthesis of bioactive compounds and other molecules that positively affect human health and also modify the physicochemical and sensory characteristics. Owing to the complexity in nutrients in milk, LAB are known to “turn on” a standard set of genes that generate enzymatic activities well identified in fermented products so far. However, little is known about the metabolic potential of LAB when grown in minimal medium or the effect on lipolytic activity specifically toward milk phospholipids (PL). In this work, we aimed to evaluate the growth of different LAB strains from our culture collection (OSU library) in a minimal medium (glucose and tryptone modified) added with different concentrations of PL (0, 0.5 and 1% wt/vol) to determine lipolysis and its sub-products. The biological effect of the resulting metabolites on intestinal cells was further evaluated measuring absorption to Caco-2 cell. After screening 95 different strains of LAB we have found that L. casei, L. pentosus, L. plantarum, P. acidilactici and P. lolli are able to grow in a medium enriched with an optimized concentration of milk PL (0.5% wt/vol) producing an interesting mixture of metabolites (not identified peaks by HPLC-MS). Even though the growth rate decreased with the higher concentration of milk PL, as well as biomass production, the lipolytic activity was greatly increased as determined in vitro using 4-nitrophenyl acetate and 4-nitrophenyl octanoate, and α-naphtyl-acetate as substrate in zymogram experiments. We also isolated a protein with an approximate mass of 40 kDa, evaluated by SDS-PAGE, which is responsible for the predominant lipolysis observed by zymography. This enzyme product on PL modification or fragmentation represents a key factor that affects cell culture response as shown by our preliminary Caco-2 cell trials.