Buttermilk is rich in milk fat globule membrane (MFGM), which consist of a well-defined group of milk phospholipids (MP). MP play an important role in brain and nervous system development. However, intact MP are also known for its low absorption in the intestine when compared with glycerolipids. Lactic acid bacteria (LAB) is one of the most common bacteria used in dairy products. Many of the LAB are beneficial for health, improving gut function, and regulating immune response. Moreover, the metabolites: the lipases and proteases produced by them can hydrolysate lipids and proteins, which can lead to better absorption of nutrients. Studies showed that metabolites produced by LAB are associated with increased absorption of sphingomyelin and potentially all the MP. The hypothesis is that LAB with lipolytic activity resulting from buttermilk fermentation with MP enriched can increase the presence of enzymes that can modify phospholipid and produce phospholipid hydrolysates with presumptive higher absorption. Bacteria characterization, classification and isolation were performed from the collection at OSU in Jimenez’ laboratory using 16S rDNA sequencing. Quantitative lipolysis and proteolytic activity were tested using α -naphthyl acetate, 4-nitrophenyl derivatives of C2-C14 and azocasien. Seven promising strains, one negative control, Staphylococcus warneri, and one positive control, Enterococcus faecalis were tested to screen their functional characteristics. Testing includes resistance to digestion including resisting low pH and bile salts, high values in auto-aggregation and hydrophobicity, and antimicrobial activity against indicator pathogenic strains: Escherichia coli ATCC 25922 and Listeria innocua ATCC 51742. Furthermore, antibiotics resistance was tested using eight antibiotics: chloramphenicol, vancomycin, tetracycline, erythromycin, ampicillin, kanamycin, clindamycin, and streptomycin. Virulence factors (agg, ace, asa1, fbp, cbp, mazm, eFaA, hdc, odc, tdc, gelE, hyl, esp and cyt) were screened by polymerase chain reaction (PCR). Three strains were selected (Lactobacillus casei 2, Lactobacillus helveticus 57B and, Lactobacillus acidophilus 70A) to continue in fermentation experiments. Selected strains were first grown in minimum medium to ensure their lipolysis ability in simple medium, then grown in 10% (w/v) skim milk (as control) and compared to those grown in 10% (w/v) buttermilk with or without 0.5% of MP added. Lipolysis products were analyzed by TLC, and HPLC. A lipolytic enzyme was detected using native protein gel: zymogram technique and Coomassie blue staining. Protein bands stained with Coomassie blue were cut and sent to amino acid/peptide sequencing by LC-MS/MS to identify the enzyme. Most of the LAB strains could digest lipid and protein substrate. The three selected LAB strains showed to have better functional properties than the commercial probiotic control: E. faecalis, including resistance to digestion and the antibiotic resistance and virulence factor safety tests. The analysis results suggested LAB strains behave differently when fermented in different media. LAB strains can break down the MP and produce MP hydrolysate when fermented with a low concentration (0.015%) of the MP. The LAB strains showed higher ability in producing lipolytic enzyme and phospholipid hydrolysate with additional MP. Buttermilk also proved to have more available MP for LAB strains utilization than skim milk. In conclusion, Lactobacillus strains were able to hydrolysate complex phospholipids producing lipolytic enzyme and formed phospholipid hydrolysates within one day of fermentation. Data also demonstrated LAB strains could help with digestion of phospholipids, and other lipid and protein substrates. The results of this work can be used to increase the bioavailability of MP and other dairy nutrients and their application in fortified dairy products and pharmaceuticals.